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Category: Immunology
Manual of Molecular and Clinical Laboratory Immunology is now available on Wiley.comMembers, use the code ASM20 at check out to receive your 20% discount.
THE authoritative guide for clinical laboratory immunology
For over 40 years the Manual of Molecular and Clinical Laboratory Immunology has served as the premier guide for the clinical immunology laboratory. Led by Editor-in-Chief, Barbara Detrick, the Manual has reflected the exponential growth in the field of immunology over the past decades from basic serology testing to the present wide range of molecular analyses. This eighth edition reflects the latest advances and developments in the diagnosis and treatment of patients with infectious and immune-mediated disorders.
The Manual features detailed descriptions of general and specific methodologies, placing special focus on the interpretation of laboratory findings, and covers the immunology of infectious diseases, including specific pathogens, as well as the full range of autoimmune and immunodeficiency diseases, cancer, and transplantation.
Written to guide the laboratory director, the Manual will also appeal to other laboratory scientists, especially those working in clinical immunology laboratories, and pathologists. It is also a useful reference for physicians, mid-level providers, medical students, and allied health students with an interest in the role that immunology plays in the clinical laboratory.
Hardcover, 1,340 pages, full-color illustrations, index.
The “-omics” revolution has begun. Since the publication of the previous edition of Manual of Molecular and Clinical Laboratory Immunology, there have been major technological advances that are facilitating the application of genomics, proteomics, and microbiomics to better understand human health and disease. These disciplines are truly complementary, as genomics is directly linked to proteomics, and these new technologies are providing an improved understanding of the functional consequences related to alterations in the genome that lead to changes in the protein products of genes. The major advancements in next-generation sequencing have made characterization of the human microbiome a reality. Major efforts in microbiomics currently are focused on evaluation of the human bacterial community in health and disease. These three disciplines find common ground in the field of immunology, in which the genome provides the triggering instructions for protein products that are critically important for immune function and host defense. Alterations in the genetic code are now known to contribute to an ever-growing list of defects in immune function that result in susceptibility to microbial disease. In addition, altered immune function leading to inflammatory disease will be further clarified with these powerful new tools. Early lessons in the study of the human microbiota have revealed that commensal bacteria interact directly with the immune system to aid and model immunity. They also provide advantage to the host in settings of health and contribute to pathogenesis in circumstances of disease. The future of clinical immunology at a diagnostic and therapeutic level increasingly will rely on these techniques and further advances that will evolve more and more rapidly.
Disorders of the immune system affect a significant number of individuals, with prevalence estimates (per 100,000) determined by registries ranging from 5.6 in Australia (1) to 4.979 in France, 2.6 in The Netherlands, and down to 1.377 in Germany (2). Accurate diagnosis of immune system disorders may allow early intervention prior to extensive illness for severe disease, or more specific treatment in the case of later-diagnosed or milder disease. This chapter explores some of the current molecular methodologies for detecting disorders of the immune system and highlights specific pitfalls that may hinder accurate diagnosis.
Humans, in many respects, are not single organisms, but more like coral reefs, complex assemblages of myriad diverse creatures. The human microbiota, the bugs living in association with humans, contains more cells than comprise the human body itself (1). Like the fish on a reef, the microbes interact with each other, sometimes competing and sometimes collaborating, to form a complex and resilient food web. Some of the population promote the growth and development of the reef, while others consume the reef material, turning the hard corals to sand.
The diagnostic immunology laboratory relies heavily on protein measurements, especially with the explosion of clinically relevant biomarker analysis. Of particular import are the tremendous advances that have been made in the technology for protein detection, and while not all of it has gained traction in the clinical immunology laboratory, this remains an area of huge growth. However, regulatory processes have not kept up with the burgeoning research in the area of protein analysis, and new diagnostic tests for protein analytes are approved for clinical testing at a glacial pace. Nonetheless, it is critical for the clinical immunologist to understand these advances and determine how they can best be utilized in the clinical laboratory. Besides keeping pace with the rapidly changing technology, the age-old fundamental principles of analytical validation of new tests, protein based or not, are still applicable. This chapter covers the basic principles of protein testing in the clinical laboratory and provides special emphasis on the role of mass spectrometry (MS) in diagnostic protein analysis.
The section on immunoglobulin methods covers the basic genetic background for immunoglobulin production, measurement of immunoglobulins, identification of monoclonal protein products by serum protein electrophoresis and immunofixation, and detection of oligoclonal bands in cerebrospinal fluid. In addition, this section covers the characterization of cryoglobulins and cryofibrinogens and an overall strategy for using all the techniques in this section to detect, stratify risks of progression of, and monitor patients with monoclonal gammopathies.
Immunoglobulins are a heterogeneous group of glycoproteins produced by B lymphocytes and plasma cells. A single person can synthesize 10 million to 100 million different immunoglobulin molecules, each having distinct antigen-binding specificities. This great diversity in the so-called humoral immune system allows us to generate antibodies specific for a variety of substances, including synthetic molecules not naturally present in our environment. Despite the diversity in the specificities of antibody molecules, the binding of an antibody to an antigen initiates a limited series of biologically important effector functions, such as complement activation and/or adherence of the immune complex to receptors on leukocytes (1). Resolution of the immunoglobulin structure has revealed how these molecules can have such great diversity in antigen-binding activities while maintaining conserved effector functions, such as complement activation.
Quantification of intact serum immunoglobulins has proven useful in the evaluation of patients with suspected immunodeficiency disorders, lymphocyte and plasma cell neoplastic diseases, allergic conditions, and some chronic inflammatory and autoimmune disorders. Since the advent of quantitative immunoglobulin assays nearly 50 years ago (1), increasingly robust analytical methods have been developed. The armamentarium of intact immunoglobulin assays was first substantively expanded by the development of immunoglobulin light-chain measurements in which the light chains are bound to heavy chains (intact immunoglobulins). The last decade has seen the development of both free (unbound) immunoglobulin light-chain assays and heavy/light-chain (HLC) or junctional epitope assays, the latter of which allows for individual measurements of IgGκ, IgGλ, IgAκ, IgAλ, IgMκ, and IgMλ (2, 3). Despite these advances in immunoglobulin and light-chain quantification methods, there remain technical complexities that can influence accuracy and, in turn, proper clinical application. Equally important, even with robust assays, is the need for thorough understanding of the clinical indications for, and limitations to, immunoglobulin and related measurements.
Protein electrophoresis is performed on serum (SPEP) and urine (UPEP) to detect and quantify monoclonal gammopathies (M proteins). In effect, serum and urine protein electrophoresis provides an immunochemical biopsy of the humoral immune system. However, other clinically relevant information is also available from examination of the proteins demonstrated from these studies. This chapter reviews basic principles of electrophoresis, the types of apparatus that are available, quality control and quality assurance procedures, costs, and illustrative patterns with recommended interpretations.
The characterization of immunoglobulins spans a spectrum of methods, including molecular analysis of gene usage and rearrangement, quantitation of immunoglobulin heavy chains as well as intact and free light chains, qualitative assessment and characterization of clonality, and identification of abnormalities that may be clinically significant, such as hyperviscosity syndrome, cryoglobulinemia, and amyloidosis (AL). This chapter focuses on qualitative methods for the assessment and characterization of clonality. The methods include agarose gel electrophoresis (AGE) with immunofixation, capillary electrophoresis (CE) with immunosubtraction (ISUB), and isoelectric focusing with immunoblotting or immunofixation. All three methods can be used to identify monoclonal, oligoclonal, and polyclonal immunoglobulin populations and to identify the heavy and/or light chains contained in the population. Immunofixation electrophoresis (IFE) and ISUB electrophoresis are diagnostic tools used for the identification of monoclonal gammopathies and, conversely, for the confirmation of polyclonal hypergammaglobulinemia. Isoelectric focusing with immunoblotting or immunofixation is a cerebrospinal fluid (CSF) diagnostic test for the identification of oligoclonal bands in multiple sclerosis (MS).
Cryoglobulinemia is one of a group of syndromes characterized by the induction of clinical and/or laboratory abnormalities by cold. Cryoglobulins are immunoglobulins (Igs) that precipitate out of solution below core body temperatures, either as a single isotype (simple cryoglobulins) or as immune complexes in which both antibody and antigen are Igs (mixed cryoglobulins). In some instances, cryoglobulinemia may coexist with other related but usually distinct forms of cold hypersensitivity, such as Raynaud's phenomenon, cold agglutinin activity, or cold-dependent activation of complement (CDAC) (1).
The preceding chapters in this section have dealt with several aspects of serum and urine protein analysis for immunoglobulins. Overwhelmingly, the single most important reason for performing these studies is to detect monoclonal gammopathies. This chapter briefly summarizes the various plasma cell proliferative diseases and then describes the use of the electrophoretic and nephelometric assays for the diagnosis, prognosis, and monitoring of monoclonal gammopathies. There are some general strategies for all laboratories as well as specific tests that are needed, depending on the disease presentation.
The field of complementology has advanced remarkably in the past 2 decades, due to newly found connections between often subtle complement abnormalities and diverse diseases. This is reflected in the exponential growth of new publications and the development of novel therapeutics that were previously unavailable for treatment of patients with complement deficiencies or other abnormalities of the system (1). The interest in complement displayed by the pharmaceutical industry has been motivated not only by the desire to create therapeutics for patients with rare complement-mediated diseases but by the necessity to prevent undue adverse events caused by complement activation when new drugs or delivery systems are used in vivo. One of the first examples of this in humans was the development of an anaphylactoid response following the infusion of radiocontrast agents or exposure of blood to some of the early types of dialysis membranes that activated complement (2). Additional examples of complement activation by diverse compounds include liposomes, nanoparticles of various types, biologicals (mainly antibody based), and DNA- or RNA-based drugs such as phosphorothioate oligonucleotides (antisense) (3, 4). The term complement activation-related pseudoallergy, or CARPA, applies to some of these reactions (5).
Complement evolved in parallel with coagulation as part of the primordial explosion of life in the Cambrian era. Remnants of this connection still exist in vertebrate animals as well as invertebrates. A classic example is the horseshoe crab, Limulus polyphemus, still sought after by scientists today to test substances for traces of bacterial lipopolysaccharide (endotoxin). The system used by the crabs had a molecule that recognized bacteria or other invaders that got into the hemolymph of the crab. An enzyme triggered by the first molecule induced local coagulation that served to trap the microbes so that they could be destroyed by phagocytes in the animal's circulation.
The innate immune system has traditionally been described as the first line of the body's defense against invasive pathogens. Such a response then leads to an inflammatory response which may also include coagulation. Activation of the innate immune response is mediated by pattern recognition molecules, which may be membrane-bound (e.g., cell-associated Toll-like receptors, NOD-like receptors, and RIG-I-like receptors) or soluble proteins. Recognition of foreign or altered structures in the body by some of the soluble pattern recognition receptors may lead to activation of the complement system and thus trigger one of the innate antimicrobial defense mechanisms. Such complement-activating soluble pattern recognition molecules include the collectins (lectins, i.e., carbohydrate-binding proteins, that use a collagen helix for stabilization of the molecule), i.e., mannan-binding lectin (MBL, also known as mannose-binding lectin), collectin K1 (CL-K1), and collectin L1 (CL-L1), and the ficolins (proteins that contain a fibrinogen-like domain and use a collagen helix for stabilization of the molecule), i.e., H-ficolin, L-ficolin, and M-ficolin (1, 2).
The complement system is the cornerstone of innate immunity. As one of the first lines of host defense, it plays a major role in microbial killing, immune complex handling, apoptotic cell clearance, tissue homeostasis, and modulation of adaptive immunity (1–3). Critical to these functions is the sequential triggering of a series of cascades that result in the generation of metastable protease complexes and culminate in the formation of membrane attack complex (MAC) (4). Improper regulation of these cascades is associated with the development of multiple different diseases. In this chapter, we focus on the clinical consequence of dysregulation of the alternative pathway (AP) of complement. We first provide a review of the AP and then illustrate the consequence of its dysregulation by describing two ultrarare diseases: atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G).
Since the last publication of the Manual of Molecular and Clinical Laboratory Immunology almost a decade ago, developments in computer technology, digital electronics, and laser and fluorochrome chemistry have led to the manufacture, adoption, and common availability of clinical flow cytometers with the capacity to routinely analyze 10 (or more) parameters simultaneously. This “polychromatic flow cytometry” capability requires significant expertise in all phases of clinical flow cytometry, from antibody panel design and sample preparation to cytometer setup and acquisition and, finally, the complex task of analyzing highly parametric data files. The first chapter of this section, from Philip McCoy's laboratory at the National Institutes of Health, Bethesda, MD, provides a comprehensive overview of the technology, sample preparation, quality control, and analytical challenges involved in the design and analysis of polychromatic flow cytometry. This chapter provides insight into the considerations required for the design and analysis of highly parametric flow cytometry assays and alerts the reader to the quality control and quality assurance practices necessary to ensure consistent and reliable polychromatic flow cytometry assays.
For nearly half a century, flow cytometry has been a tool used by biologists to study features of individual cells in a rapid and unbiased manner. The measurement of cellular features has relied upon intrinsic properties of the cells which can be examined by laser-light scattering or by the addition of extrinsic fluorescent probes, such as dyes or fluorochrome-conjugated antibodies that can make cellular features quantifiable. The early flow cytometers relied on only one or two lasers, or a mercury arc bulb, for excitation light, and generally measured only one fluorescent parameter. In the late 1970s, Stohr (1) and Dean and Pinkel (2) described dual-laser excitation for flow cytometry, which permitted the simultaneous use of two spectrally distinct fluorochromes, signaling the genesis of polychromatic flow cytometry. At roughly the same time, Loken and colleagues proposed a method to overcome the difficulties caused by the spectral overlap of fluorochromes excited off a single laser—a method we now refer to as “compensation” (3). These developments laid the foundation for future high-polychromatic flow cytometry, as the bases for both multi-laser excitations as well as exciting multiple fluorochromes off each laser had now been described.
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening acquired hematopoietic stem cell disorder resulting from the somatic mutation of the X-linked phosphatidylinositol-glycan complementation class A (PIG-A) gene (1–4). In normal individuals, this gene encodes an enzyme involved in the first stage of glycophosphatidylinositol (GPI) biosynthesis. In PNH, as a result of the mutation(s) in the PIG-A gene, there is a partial or absolute inability to make GPI-anchored proteins, including complement-defense structures such as CD55 and CD59 on red blood cells (RBCs) and white blood cells (WBCs) (5–8). Absence of CD59 in particular (9, 10) and CD55 on RBCs is largely responsible for intravascular hemolysis associated with clinical PNH (reviewed in reference 11).
The pioneering studies of Thomas et al. in the late 1950s first established that a cellular component of syngeneic bone marrow was capable of regenerating multilineage hematopoiesis in cancer patients receiving supralethal doses of radiation (1, 2). For the next 20 or more years, the majority of autologous and allogeneic hematopoietic stem/progenitor cell transplants were performed utilizing bone marrow as a source of stem cells (reviewed in reference 3). Circulating stem cells were also detectable in steady-state peripheral blood but were extremely rare, as evidenced by their low plating efficiency relative to marrow leukocytes in early colony-forming cell assays (4). Although blood stem/progenitor cells could be collected from steady-state peripheral blood by leukapheresis, the number of procedures required to obtain sufficient cells for transplant initially precluded their widespread use (5). With the development of more sophisticated colony-forming cell assays, a variety of reports in the mid-1980s clearly demonstrated the feasibility of obtaining clinically useful numbers of peripheral blood stem/progenitor cells from cancer patients recovering from chemotherapy (6–8). The availability of a number of hematopoietic cytokines used either singly or in combination with other cytokines and/or chemotherapy (9) has facilitated the harvesting of peripheral blood stem cells (PBSC) to the point where it is a preferred alternative to marrow for autologous and, increasingly, allogeneic transplantation (reviewed in reference 10).
Primary immunodeficiency diseases represent an extremely diverse group of disorders caused by mutations in genes that code for multiple components of both the innate and adaptive immune systems. These mutations adversely affect immune homeostasis ultimately leading to increased susceptibility to infections, autoinflammatory disorders, and other symptoms of immune dysregulation. Although there are now more than 200 diseases that have been officially classified (1), diagnosis of primary immunodeficiency remains challenging and is often delayed due to the extremely broad range of signs and symptoms, variations in the severity and range of symptoms at presentation, and the overlap of presenting clinical symptoms of immunodeficiency with the clinical signs and symptoms of common illnesses. The extremely broad and large number of genetic abnormalities that make up the primary immunodeficiency disorders would suggest that DNA sequencing of the exome or genome should be the ultimate diagnostic modality. A recently published article describes how such an approach (i.e., DNA sequencing of the whole exome) resulted in an increased ability to detect genes associated with both known and new immune disorders (2).
Acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma are malignant neoplasms of progenitor cells committed to B-lineage or T-lineage lymphopoiesis, termed lymphoblasts (1). Distinction between lymphoblastic leukemia (predominantly bone marrow with or without peripheral blood involvement) and lymphoblastic lymphoma (predominantly mass-forming nodal and/or extranodal disease) is largely clinical; however, the presence of greater than 25% bone marrow blasts generally prompts the designation of “leukemia” in most protocols, regardless of the presence of a mass lesion (2, 3). In the most recent World Health Organization (WHO) classification (2–4), B lymphoblastic leukemia/lymphoma is further subclassified on the basis of recurrent genetic abnormalities, while T lymphoblastic leukemia/lymphoma, despite the existence of recurrent genetic abnormalities, is not further subclassified (Table 1).
Flow cytometry is an integral tool in both the diagnosis and posttherapy evaluation of acute myeloid leukemia (AML). The strength of the technology is its capacity for rapid, sequential single-cell analysis with simultaneous evaluation of multiple antigens, thus providing a comprehensive immunophenotype for discrete cellular subpopulations. As a result, it has become the methodology of choice for determining blast lineage and immunophenotype. Cytochemical or immunohistochemical evaluation, although useful and even required in some settings, has been surpassed by flow cytometry for the evaluation of blood and marrow in most instances (1, 2). Nevertheless, flow cytometric findings must be used in conjunction with morphology, molecular, and cytogenetic findings for the complete diagnosis and subclassification of AML. Evaluation of posttherapy samples for residual acute myeloid leukemia allows enumeration of blasts as low as 1% by morphology; however, distinguishing normal or regenerating progenitors from leukemic blasts rarely can be performed by morphologic examination alone. Flow cytometry not only allows a more sensitive assay, in some cases reaching a sensitivity of 0.01% (3–5), it is also able to identify aberrancies on the leukemic blast population that allow discrimination from normal or regenerating progenitors (2, 6, 7). These attributes make flow cytometry an ideal method for evaluating for minimal residual disease (MRD) in the posttherapy setting.
Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a neoplasm of mature B lymphocytes involving peripheral blood (PB), bone marrow (BM), spleen, and lymph nodes (LN). CLL is the most common lymphoproliferative disorder in western countries and is primarily a disease of adults, often occurring during or after middle age. The diagnosis is established by blood counts, blood smears, and immunophenotyping by flow cytometry (FC) of circulating B lymphocytes (1). Although usually an indolent disease, some patients have a more rapid disease progression and require treatment earlier. Survival in patients varies from 1 year to 20 years with an 82% 5-year survival rate (2). FC demonstration of the typical CLL immunophenotype is vital for diagnosis. The differential diagnosis of CLL/SLL would primarily include monoclonal B-cell lymphocytosis (MBL) and mantle cell lymphoma (MCL), although B-prolymphocytic leukemia, marginal zone lymphoma, diffuse large B-cell lymphoma, and lymphoplasmacytic lymphoma can on occasion have some features of CLL/SLL (Table 1) (3).
Clonal plasma cell disorders (PCDs) encompass a heterogeneous group of distinct entities characterized in common by a clonal expansion and accumulation of plasma cells (PCs) in the bone marrow (BM) and/or other tissues, which is associated in the vast majority of cases with the presence of their product(s) (monoclonal immunoglobulin [Ig], M component) at detectable amounts in serum or urine (1). Although the so-called diseases of immunoglobulin deposits (e.g., primary light chain [AL] amyloidosis) and lymphoplasmacytic lymphoma (e.g., Waldenström macroglobulinemia) also belong to this heterogeneous group of disorders, its most representative diagnostic subtypes include monoclonal gammopathy of undetermined significance (MGUS), solitary plasmacytoma, multiple myeloma (MM), plasma cell leukemia (PCL), and several subvariants of these entities (1, 2) (Table 1). The last four diagnostic categories of PCD are the main focus of this chapter.
The past several years have seen a rapid evolution of flow cytometry technology, moving toward more parameters with the development of instruments containing smaller and less expensive lasers and with the discovery of new dyes that extend the range of emissions that can be detected by those lasers. This is exemplified by the emergence of violet diode lasers (1) and associated quantum dot nanocrystals (2) and brilliant violet dyes (3) that are excited by violet (and most recently by UV) lasers. These developments make flow cytometry in the range of 15 or so parameters more practical than ever.
A number of remarkable and seminal advances in basic immunology, molecular biology, cellular physiology, and molecular diagnosis have led to the development of new and unique assays. These techniques, in many instances, have been applied to both research and clinical laboratories. They afford unique sensitivity and specificity for a panoply of diagnostic laboratory procedures. This section of the Manual focuses on the laboratory-based assays related to functional cellular assays and their application to clinical diagnoses (Fig. 1). The chapters in this section include major aspects of these assays.
The utility of cryopreserved peripheral blood mononuclear cells (PBMC) in clinical and diagnostic immunology is widely recognized. The use of cryopreserved PBMC offers multiple advantages for in vitro studies. The ability to batch specimens permits significant cost reductions through efficient utilization of labor and reagents and permits testing of multiple samples in a single run, thus avoiding interassay variability and providing more meaningful comparisons in longitudinal studies.
Measurement of lymphocyte activation is a key diagnostic component in the work-up of several immunological diseases: either primary in nature or altered as a result of immune-modifying therapies or other diseases that are not genetic immune deficiencies. There are many ways to measure lymphocyte activation, but the two main approaches in the diagnostic immunology laboratory include immunophenotyping (measurement of cell surface or intracellular activation markers after lymphocyte stimulation) and functional assays (where there is direct assessment of a certain function, e.g., proliferation after lymphocyte activation or cytotoxicity). Among the lymphocyte subsets that are typically assessed for activation status as a measure of immune competence, T cells are the most common, though in some contexts it is also appropriate to evaluate B cell and NK cell activation or function. This chapter focuses primarily on discussing assays and methods related to measurement of T cell activation and function.
The primary cells of the adaptive immune system are T cells, B cells, and natural killer cells. These lymphocytes assist the host in eliminating both intracellular pathogens (T cells and NK cells) and extracellular pathogens (B cells) through B cell-T cell interactions, as well as interactions with other cells and molecules of the innate immune system. B cells recognize foreign antigen by the B-cell receptor (BCR), a membrane-bound immunoglobulin generated through a complex genetic recombination process (1). The BCR recognizes conformational protein antigens as well as nonprotein antigens. Two types of B cells have been described based on expression of cell surface molecules and function. B1 (CD5+) B cells are thought to be a more “natural” type of B cell which respond to T-cell-independent forms of antigen (i.e., bacterial polysaccharides) (2, 3). B2 B cells respond to T-cell-dependent antigens, such as the classic protein antigens tetanus and diphtheria toxoids. Both classes of B cells respond to BCR binding of antigen by proliferation, differentiation into antibody-secreting plasma cells, and formation of memory B cells.
CD3+CD8+TcR αβ thymus-derived T cells form the major effector cell component of the immune response against intracellular pathogens. Naive CD8+ T cells encounter antigen, expand, and divide, and effector cells mediate a range of functions. These include major histocompatibility complex (MHC) class I restricted killing of infected host cells, noncytolytic suppression, and release of soluble cytokines and chemokines. After the acute infection is contained the number of active effector cells declines, but antigen-specific memory CD8+ T cells persist, ready to expand rapidly upon antigen re-exposure. Most CD8+ antigen-specific T cells recognize viral antigen as a peptide presented by an MHC class I molecule. The T-cell receptor of the antigen-specific CD8+ cell interacts with the MHC class I antigenic peptide complex on the surface of the antigen-presenting cell (1).
Regulatory T cells (Treg) are a small subset (<5%) of circulating CD4+ T cells. Due to their ability to suppress functions of other lymphocytes, Treg are responsible for maintaining immune responses in balance. This system of immune checks and balances exists to protect us from autoimmune diseases, prevent tissue damage resulting from intense inflammatory responses induced by infectious or non-infectious injuries and to contain chronic inflammatory reactions that might promote tumor development (1). Treg seem to be capable of fulfilling all these functions, largely due to their plasticity, which allows them to readily adjust to conditions in the local microenvironment (2). Viewed in this context, Treg can be considered as a protective mechanism designed to limit inflammatory tissue damage, but also as a potentially dangerous suppressor of immune responses that benefit the host. For example, in chronic viral infections such as HIV-1 infections, Treg could either suppress excessive immune activation, thus benefiting the host, or limit antiviral immunity needed by the host to contain the infection. Today, the role of Treg in HIV-1 remains highly controversial (3). In cancer, Treg are emerging both as contributors to cancer progression, because of their ability to block anti-tumor immune responses, and also as inhibitors of cancer progression via their ability to suppress cancer-promoting inflammation (4). It is unclear whether the anti-inflammatory activity of Treg or their suppression of antitumor immunity contributes to disease outcome, and the mechanisms responsible for regulation of Treg functions remain unknown. Factors that govern Treg behavior in situ are being intensely investigated in various human diseases. An excess of Treg (e.g., in some cancers) or their numerical or functional deficiency (e.g., in autoimmune diseases) is associated with clinical symptoms and may predict outcome. For these reasons, measurements of Treg frequency and function in the circulation, and especially in tissues, are of great importance.
Natural killer (NK) cells are a subset of innate lymphocytes initially identified for their ability to specifically kill virally infected and transformed cells without prior antigen sensitization. NK cells efficiently kill target cells through directed release of perforin-containing secretory lysosomes, a feature shared with cytotoxic T lymphocytes (CTLs) (1). Despite similarities in effector mechanisms, the strategies employed by NK cells and CTLs for target cell recognition are distinct yet complementary with respect to immune defense. Whereas CTLs express recombined, clonally distributed antigen receptors that dictate their activation and are selected for recognition of cells presenting nonself peptides in the context of major histocompatibility (MHC) class I molecules, NK cells rely on dynamic integration of signals from various germ line-encoded receptors for target cell discrimination. NK cells express numerous inhibitory receptors to detect normal expression of MHC class I and can selectively kill target cells that downregulate these molecules (2). NK cell activation by target cells with low MHC class I levels does not occur by default, but rather is mediated through engagement of different activating receptors (3). Representing an effector arm of humoral immunity, NK cells express the low-affinity Fc receptor CD16, which facilitates antibody-dependent cellular cytotoxicity (4). Moreover, supporting first-line defense against virally infected or stressed cells, a multiplicity of activating receptors that participate in natural cytotoxicity have been identified. In general, engagement of each such receptor alone is not sufficient to induce NK cell cytotoxicity. However, certain combinations of receptor signals can synergistically activate NK cell effector functions (5). Reflecting the expression of several activating receptors on NK cells that bind ligands exclusively expressed on hematopoietic cells, the ability of NK cells to kill autologous, activated immune cells is increasingly appreciated as an important immunoregulatory mechanism to control and shape adaptive immune responses (6). Upon activation, NK cells not only release granules but also abundantly produce chemokines and cytokines (7).
Chronic granulomatous disease (CGD) is a rare genetic disease (~1 in 200,000 in the U.S.) first described in the 1950s (1). It is characterized by a failure of phagocytes (polymorphonuclear neutrophils [PMN], monocytes, macrophages, and eosinophils) to generate superoxide (O2·−) and other related reactive oxygen species (ROS), leading to recurrent infections, granulomatous complications, and premature death. Generation of O2·− requires the assembly and activation of a multicomponent enzyme, NADPH oxidase (NOX2) or phagocyte oxidase (phox), a complex consisting of numerous cytosolic proteins, including p47phox (2), p67phox (2), and p40phox (3), and two membrane proteins, p22phox and gp91phox, that constitute cytochrome b 558 (4, 5). NOX2 catalyzes the reduction of molecular O2 to O2·− using NADPH generated by the oxidation of glucose through the pentose-phosphate pathway. O2·− is converted to H2O2 either spontaneously or enzymatically. H2O2 and O2·− can react to form the highly reactive hydroxyl radical, OH•. The molecular defect in CGD results from mutations in any one of 5 protein subunits of NOX2, that include gp91phox (~70% of patients), p47phox (~25%), p22phox (<5%), p67phox (<5%), and p40phox (one case identified). Because the molecular defect in CGD is the inability to generate ROS, most of the assays used in the diagnosis of CGD that are described below are based on assessments of ROS production using different probes and different detection platforms. The last two assays—flow cytometric analysis of NOX2 expression and immunoblot analysis of phox subunits—focus on identifying the specific protein defect and defining the target for genetic sequencing.
Nearly 60 years ago, the first cytokine was described. Today, >300 cytokines, chemokines, and adhesion molecules have been identified. We have witnessed an explosion in knowledge about cytokine biology. This can be highlighted by reviewing the interferon (IFN) molecules as an example. IFN was first identified in 1957 as a potent antiviral molecule. We now know that there are four major types of IFNs (alpha, beta, gamma, and lambda). Although IFN was first identified as an antiviral protein, IFNs are now recognized as critical immunoregulatory proteins capable of altering various cellular processes, including cell growth, differentiation, gene transcription, and translation. The advent of innate immunity and Toll-like receptors revealed the intimate role of IFNs in innate immune responses. The presence of IFN-α is a critical component of the autoimmune disease systemic lupus erythematosus. Advances in biotechnology and molecular biology have generated highly specific reagents with clinical relevance. In fact, the IFNs have been approved by the Food and Drug Administration for the treatment of infections, malignancies, autoimmunity, and immunodeficiency. IFN-α treatment is efficacious for hepatitis C infection, IFN-β for multiple sclerosis, and IFN-γ for chronic granulomatous disease. The examples outlined for the IFNs can be replayed throughout the cytokine kingdom.
Cytokines are low-molecular-weight proteins representing important components of inflammatory and immune reactions (1). In response to multiple stimuli (2), cytokines are rapidly induced and secreted into the extracellular milieu. However, in some situations, cytokines are constitutively present. Cytokines exert numerous biological activities which are critical for host defense, physiologic responses to stress, and immune surveillance. Cytokines, along with complement, are considered to be part of the innate immune system. The world of cytokine biology has exploded in the past decades. It can be said without hyperbole that cytokines are critical from birth (gestation) (3) to death (apoptosis) (4).
In the years since intracellular cytokine staining (ICS) was first developed (1), this method has become a standard way to analyze the functions of immune cells by flow cytometry. It has been used with specific antigen stimulation (2, 3) to read out T cell populations responsive to vaccines (4–6), pathogens (7, 8), cancer (9–11), or allergens (12), in both human and animal models. The technique has been combined with MHC-peptide multimer staining (11, 13), including combinatorial tetramer staining that allows the interrogation of many epitope specificities at once (14, 15). Combined assays with tetramers can be done by first staining with tetramer(s), then stimulating with a nonspecific agent such as PMA+ionomycin, which bypasses the tetramer-occupied T cell receptors. ICS has also increasingly been used with detection of CD154 (16, 17) or CD107a (18) induction, as markers of activation and degranulation, respectively. When used with cell surface and other intracellular phenotyping markers, such multiparameter ICS assays are a tool of choice for functional characterization of T cells and other cytokine-producing cell populations. ICS has also successfully been converted to the mass cytometry platform (19) allowing the use of many more simultaneous markers without significant spillover between readouts.
The active movement of leukocytes towards a site of antigen challenge, infection, or tissue damage represents a central aspect of the establishment of both inflammatory and immune responses (1–4). The movement of cells towards a chemical gradient of a particular stimulus or chemotactic factor is called chemotaxis. Chemotactic factors that induce the directional movement of leukocytes include the chemokines, a super family of proteins 8 to 10 kDa in size that signal chemotaxis through seven transmembrane G protein-coupled cell surface receptors (GPCRs) (1, 2). In this chapter, the methodological approaches to studying the role of chemokines and chemokine receptors in the physiology of immune and inflammatory responses are described. Although assays of chemokines or chemokine receptors have yet to be used for widespread clinical applications, this chapter briefly reviews the role of these proteins in the pathophysiology of several inflammatory diseases and illustrates potential clinical settings in which measuring these proteins or studying their functional activity may be of useful.
Many cytokines can be measured in the circulation via antibody-based detection protocols such as enzyme-linked immunosorbent assays and multianalyte bead systems. These measurement systems are robust and adaptable to many different analytes, and thus the large-scale measurement of cytokines in the circulation in human populations and disease states is possible. The measurement of cytokines in the circulation may provide a unique window into the immune response and the biology of human immune-related diseases. Cytokines represent messages sent between cells of the immune system, and thus confer a great deal information about ongoing immune processes. It is attractive to think that by intercepting these messages, we may be able to gain valuable insights regarding human disease diagnosis, prognosis, and potential therapeutic avenues. In this chapter, we review some of the ways in which cytokines are applied to clinical medicine. While cytokines can also be detected in tissue, this requires a biopsy, and for the purpose of this chapter we focus our discussion on circulating cytokines.
Anticytokine autoantibodies are an emerging mechanism of disease pathogenesis that have been shown to elicit a broad range of clinical phenotypes. Some anticytokine autoantibodies can lead to immune susceptibility, with examples including nontuberculous mycobacteria, salmonellae, or fungi due to autoantibodies against gamma interferon (IFN-γ) (1); staphylococcal infection due to anti-interleukin-6 (IL-6) autoantibodies (2); Burkholderia gladioli infection due to anti-IL-12p70 autoantibodies (3); chronic mucocutaneous candidiasis due to anti-IL-17 and anti-IL-22 autoantibodies (4, 5); and cryptococcal meningitis (6) or Nocardia infection due to anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies (7). Other diseases in which immunodeficiency is not the primary presentation include the severe lung disease pulmonary alveolar proteinosis (PAP) caused by anti-GM-CSF autoantibodies; pure red cell aplasia (8); pure red-cell aplasia due to antierythropoietin autoantibodies (9); and severe osteoporosis associated with antiosteoprotegerin autoantibodies (10). For other anticytokine autoantibodies, such as anti-IFN-α autoantibodies detected in thymoma or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, their biological effect is not immediately apparent (11). Diagnosis has important implications for management because therapies can be used to directly target the underlying mechanism, i.e., a neutralizing antibody, and not just their ultimate clinical consequences, with approaches such as B-cell depletion (12–14) or receptor agonists (15).
Immunohistochemistry techniques are integral to the practice of anatomic pathology. In contrast to the 7th edition of this Manual, in which a separate chapter discusses the principles and advances of immunohistochemistry, these techniques have been directly incorporated in two chapters along with figures illustrating the detection of antibodies involved in renal and cardiac diseases. In the first chapter, Collins et al. provide a brief history of immunofluorescence. Methods for the handling, freezing, and storage of tissue specimens, typically obtained by biopsy, are discussed. While most immunofluorescence studies are performed with unfixed frozen tissue, tissue fixation and tissue sectioning are detailed. Direct immunofluorescence is illustrated for the detection of immunoglobulin G (IgG), IgA, IgM, C3, C1q, fibrinogen, and kappa and lambda light chain deposition in renal tissue. A brief discussion of dual fluorescence and microscope instrumentation is followed by an overview of interpretation. In the second chapter, Collins and Smith discuss Western blot analysis for the detection of antibodies specific for glomerular basement membrane and phospholipase A2 receptors. Special requirements are highlighted for antigen presentation, enzymatic digestion of glomeruli, and transfer onto nitrocellulose paper. Section G provides an overview of immunohistology and immunopathology measurements in present-day clinical practice.
Immunofluorescence is a well-established technique for the detection of antigens in tissue sections or cell suspensions (1, 2). The technique was developed in 1941 by Albert Coons to demonstrate the presence of pneumococcal antigens in tissue (3). Since then, immunofluorescence has become a crucial tool in the diagnosis and determination of prognoses of immunologically mediated disease (4). Direct immunofluorescence (defined as the application of specific antibodies to detect specific antigens in tissue) is a sensitive and well-established technique for the detection of tissue-bound immunoglobulins, their subclasses, complement components (C3, C1q, and C4d), amyloidogenic proteins, and fibrin-fibrinogen. In the kidney, many forms of primary glomerulonephritis are characterized by deposition of immunoreactants in distinctive diagnostic patterns. The primary renal targets are the glomerulus, the proximal tubules, and the interstitium. In the heart or open fat pad biopsy specimens, direct immunofluorescence is useful to speciate the type of amyloid identified on specimens with positive Congo red staining in paraffin-embedded tissue (5). The diagnosis of antibody-mediated rejections in heart and kidney allografts is facilitated by the detection of C4d (6–8). This chapter will also emphasize the common immunofluorescence techniques used for diagnosis and interpretation with kidney and skin biopsy specimens.
Anti-basement membrane antibodies and diffuse alveolar hemorrhage (Goodpasture's syndrome) receives its eponym from Ernest Goodpasture. While studying the influenza pandemic after World War I, Goodpasture studied an 18-year-old male, who died of pulmonary hemorrhage, and diagnosed his pathological findings as systemic vasculitis (1). Although the disease that Goodpasture described is probably different from the disease that now carries his eponym (2), Goodpasture's disease is now well recognized as one of the causes of pulmonary hemorrhage and/or acute renal failure. Immunofluorescence later identified that some cases of pulmonary hemorrhage and renal failure were associated with the linear deposition of immunoglobulins deposited along the pulmonary and glomerular basement membranes (GBM) (3–5). The term Goodpasture's is now generally reserved for the diseases of pulmonary hemorrhage and renal failure with anti-basement membrane antibodies (6, 7).
This section contains chapters describing methods for the immunologic diagnosis of acute, chronic, or latent infections and nonsuppurative sequelae caused by bacteria, rickettsiae, mycoplasmas, bartonellae, and borreliae. The majority of the methods described in these chapters measure antibodies. In situations where it is difficult to culture the infectious agent, antibody detection is one of the only effective ways to identify the infecting organism. The described methods cover a wide range of antibody assays, including hemagglutination, indirect fluorescence, complement fixation, and enzyme-linked immunosorbent assays.
Group A streptococcus, also known as Streptococcus pyogenes, is an important bacterial pathogen that is the most frequent bacterial cause of acute pharyngitis. It also causes a multitude of other cutaneous and systemic infections, including impetigo, scarlet fever, necrotizing fasciitis, and streptococcal toxic shock syndrome. The pathogen has a unique tendency to initiate autoimmunity after acute infection, resulting in the nonsuppurative sequelae acute rheumatic fever (ARF) and poststreptococcal acute glomerulonephritis (AGN). The heart disease resulting from ARF has been responsible for substantial morbidity and mortality in all parts of the world.
Helicobacter pylori is a Gram-negative, microaerophilic spiral bacterium which is recognized as the primary cause of chronic gastritis in humans. Untreated H. pylori infection precedes and is required for the development of most cases of gastric and duodenal ulcers (1, 2). Further, H. pylori infection substantially increases the risk of development of gastric cancer in some populations. Following the discovery and identification of H. pylori as a significant human pathogen, it is now recognized as probably the most common bacterial infection of humankind, infecting approximately 50% of the world's population. Specific antibiotics combined with proton pump inhibitors are now routinely used to treat and eliminate previously chronic gastroduodenal diseases. For their novel work in helping us to understand the critical role played by H. pylori in peptic ulcer disease, Robin Warren and Barry Marshall were awarded the Nobel Prize in Physiology or Medicine in 2005.
The laboratory diagnosis of syphilis has relied, in large part, upon serologic methods for over 100 years (1). The causative organism, Treponema pallidum, is not amenable to in vitro culture, and antigen detection and nucleic acid amplification techniques have not become routinely used. While many of the serologic methods in use now have been in use for decades, their application, specifically the standard testing algorithm, is changing. In order to understand the interpretation of alternative testing algorithms, a thorough understanding of test characteristics is necessary. This chapter will review serologic tests for syphilis and their interpretation when they are applied to adults with and without HIV infection and to children.
Lyme disease, relapsing fever, and leptospirosis are infections caused by spirochetes, a phylogenetically ancient and distinct group of microorganisms among the prokaryotes.
Tuberculosis (TB) is one of the most common serious bacterial infections and remains a major challenge to global health. One-third of the human population has been infected with Mycobacterium tuberculosis. Globally, drug-resistant tuberculosis is emerging as a new epidemic, with approximately 0.5 million new multidrug-resistant cases annually. TB is second only to HIV as a worldwide cause of death from an infectious disease, and HIV infection, in turn, is a major risk factor for TB.
Mycoplasmas and ureaplasmas are members of a unique group of organisms (class Mollicutes) that are characterized by their small genomes, lack of cell walls, sterols in cell membranes, and complex nutritional requirements. The role of Mycoplasma and Ureaplasma species in human diseases was largely underappreciated until recent years. As a result, most diagnostic laboratories ignored them. Because of their complex nutritional requirements as well as their adaptation to the host during infection, these fastidious organisms can be difficult and time-consuming to culture from patient samples. However, there are improved methods for detection, including PCR detection assays, serologic assays, and commercially available growth media, but these are still limited compared with the products for other organisms.
Chlamydiaceae is a unique family of bacteria which consists of small Gram-negative coccobacilli that are obligate intracytoplasmic organisms, typically causing infection in warm-blooded animals (1). The genus name Chlamydia first appeared in the literature in 1945 but was not fully recognized until 1956 (1, 2). The family had only one genus until 1999 when the genus Chlamydophila was added. However, the name Chlamydophila has not become widely adopted in the medical community, and Chlamydophila spp. are still commonly referred to as Chlamydia spp.
Organisms belonging to the families Rickettsiaceae, Anaplasmataceae, and Coxiellaceae are a diverse group of intracellular bacteria that cause a variety of infections in humans. As in the case of the Rickettsiaceae and Anaplasmataceae, hematophagous arthropod vectors play a role in maintaining the agents in nature and transmit the agents to cause disease. Coxiella burnetii, the sole pathogen of the family Coxiellaceae, is a zoonosis spread to humans by the inhalation of contaminated aerosols. In the last 2 decades, there has been an emergence of new rickettsial and ehrlichial species as well as new syndromes attributed to species previously deemed nonpathogenic. The growing list of pathogens and syndromic illnesses adds a layer of complexity to their recognition. Additionally, the clinical manifestations of infections caused by these agents are protean and nonspecific. For these reasons, establishing an accurate and timely diagnosis is often difficult. Therefore, knowledge of available laboratory tests, their interpretation, and their shortcomings is crucial for the management of infections caused by these bacteria.
The three families of bacteria described in this chapter share several common characteristics despite the phylogenetic distance between the group that includes Bartonella and Brucella, from the Alphaproteobacteria, and the more distantly related Francisella in the Gammaproteobacteria class. All three genera are zoonotic bacteria with species capable of infecting both animals and humans and are fastidious with special growth requirements; many species among these three genera cause emerging infections in humans. The diversity of natural animal reservoirs for members of the genera Bartonella and Brucella are just now becoming fully defined and appreciated and are likely all around us. Brucella spp. and Francisella spp. are well-established as agents that warrant special attention and focus because of their potential for misuse and intentional release in acts of bioterrorism or biowarfare. This chapter briefly summarizes our knowledge of the taxonomy, epidemiology, and pathobiology of these zoonotic bacteria and describes the immunologic and molecular tools for the laboratory diagnosis of infections caused by these microbes.
Combining the sections on parasitic and fungal infections into one unit stems not so much from the similarity between parasites and fungi but rather from their not being part of the infectious diseases mainstream. From a phylogenetic perspective, parasites (even the single-cell protozoa) differ from the fungi by a considerable evolutionary distance; interestingly, based on phylogenetic analyses, the differences between protozoan parasites and fungi are less significant than the differences between protozoa and helminth parasites.
Definitive diagnosis of parasitic infections is made by identification of parasites in properly collected specimens or in affected tissues. Microscopy for observation and identification of parasites is the laboratory method of choice for the diagnosis of some important parasitic infections, namely, malaria, babesiosis, and enteric parasitic infections. There are, however, important parasitic diseases that cannot be diagnosed by microscopic examination of clinical specimens. In infections such as angiostrongyliasis, schistosomiasis, paragonimiasis, and strongyloidiasis, parasites may be detected in stool or other specimens, but due to the limitations of intermittent shedding or sampling, direct observation of parasites is not sensitive or reliable. So, for these infections and others caused by parasites that are localized and sequestered in tissues, such as baylisascariasis, cysticercosis, echinococcosis, toxocariasis, toxoplasmosis, or trichinellosis, detection of specific antibodies is almost always required to confirm clinical suspicion.
Analysis of the signs and symptoms of disease, in conjunction with an evaluation of available epidemiologic information and the results of modern imaging procedures, can often provide a presumptive clinical diagnosis of a fungal infection. However, the clinical presentation of many mycotic diseases is nonspecific, and a presumptive diagnosis must therefore be confirmed by appropriate laboratory tests. A definitive diagnosis of fungal disease is usually based upon the isolation of the etiologic agent in culture and/or microscopic demonstration of the organism in histopathologic or other clinical specimens. Unfortunately, these laboratory methods are insensitive and often unsuccessful, despite repeated sampling efforts. In the absence of positive microscopy or culture, immunologic and molecular tests offer alternative laboratory procedures to aid in the diagnosis of a mycotic disease. Advances in molecular diagnostic methods and the commercial introduction of new antigen and antibody detection tests hold promise for an earlier and more specific diagnosis than previously possible. Antibody detection tests are often helpful for the diagnosis of fungal infections. For example, when acute- and convalescent-phase serum specimens are tested, seroconversion from negative to positive or a 4-fold rise in antibody titer is considered to be diagnostic.
It is probably not unreasonable to say that the clinical virology laboratory has experienced the greatest amount of change of any clinical pathology laboratory since the last edition of this Manual. The traditional virology laboratory, as found in many larger hospital-based facilities, often no longer exists, having been replaced by molecular diagnostics. Accordingly, the first chapter in this section now covers rapid and molecular diagnostic methods as well as the traditional diagnostic procedures that still pertain. Rarely are culture or antigen detection methods still in regular use. Likewise, the ability to detect low copy numbers of viral nucleic acids, as well as the ability to accurately quantify the amount of virus present based on nucleic acid amplification, has limited the practical application of serologic methods to immune status testing, with a few well-known exceptions. There are many obvious benefits associated with the adoption of molecular diagnostics for viral infections, such as an increase in the diagnostic sensitivity. In addition, these technologies allow for the rapid deployment of tests for newly described viral pathogens. Quantitative methods facilitate diagnosis and allow prognostic application and facilitate patient management. This is not to say that serologic methods have been stagnant. There has been increasing adoption of avidity-based testing for chronic viral infections to facilitate the differentiation of primary infection from reactivation.
Tremendous strides have been made in viral diagnosis in the past decade. Many infections are now treatable and, for optimum effect, antivirals must be started early. For hospitalized patients, rapid and accurate viral diagnosis is essential not only for patient management, but for infection control and prevention of nosocomial transmission. The impact of seasonal and pandemic influenza on morbidity and mortality of all ages has received increasing attention in both the medical literature and the popular press. Emerging, highly virulent viruses such as severe acute respiratory syndrome (SARS) coronavirus (CoV), Middle East respiratory syndrome CoV, and avian influenza H5N1 and H7N9 have also heightened concerns. Lastly, immunosuppressed and other vulnerable populations at risk for serious or life-threatening viral infections continue to increase. In response to these needs, there has been an explosion in viral diagnostic test development.
Human infections with herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are common throughout the world. HSV infection can cause a variety of illnesses depending on the anatomic site infected, whether it is a primary or recurrent infection, and the immune status of the person infected. Persons at risk for serious or prolonged active HSV infection are those with eczema, severe burns, or immunosuppressive conditions, such as organ transplant patients or HIV-infected persons. Primary infection occurs in individuals who have not previously been infected with either HSV-1 or HSV-2. Primary infections may be subclinical or mild enough to be unrecognized in a majority of cases, whereas clinically apparent infections comprise a wide array of presentations, ranging from mild pharyngitis or cutaneous infections to severe generalized disease and, on rare occasions, death (1). HSV-1 has a greater propensity to cause oral infections and is typically acquired during childhood. The most frequent manifestation of primary HSV-1 infection in children is gingivostomatitis. Primary HSV-1 infection in adolescents and adults usually manifests as pharyngitis or tonsillitis. Conjunctivitis, keratitis, vesicular eruptions of the skin, herpes whitlow, and encephalitis occur much less frequently. While the most common manifestation of sexually acquired HSV-2 is genital disease, both HSV-2 and HSV-1 can cause genital infection. HSV is the most common cause of genital ulcer disease in developed countries. In recent years, HSV-1 has been more frequently implicated as the etiologic agent (1). Complications of HSV-2 infection include aseptic meningitis and other neurological complications, extragenital lesions, and disseminated infection. Neonatal herpes is caused by mother-to-child transmission of HSV-1 or HSV-2 in utero, during the birth process, or during the neonatal period. Infants infected during delivery or postpartum present in one of three ways: (i) disease localized to the skin, eyes, or mouth; (ii) central nervous system disease with or without skin, eye, or mouth disease; or (iii) disseminated infection. HSV-1 seroprevalence in the United States declined about 7% from 1999 to 2004 and from 2005 to 2010, with roughly 60% of persons being HSV-1 seropositive among 40- to 49-year-olds (2). HSV-2 seroprevalence in the United States increased by more than 50% between the mid-1970s and the mid-1990s but then began to decline; in 2005 to 2010, the estimated seroprevalence was 25% in 40- to 49-year-olds (2).
Varicella-zoster virus (VZV) is a readily contagious alphaherpesvirus that commonly causes two distinct exanthematous illnesses. Primary infection with VZV causes varicella, known colloquially as chicken pox, which is characterized by a generalized vesicular rash typically accompanied by fever and other nonspecific symptoms. VZV establishes a lifelong latent infection in the dorsal root ganglia during the first infection, and it can reactivate decades later to cause zoster, also called shingles, a dermatomally distributed and often painful vesicular rash (1, 2). A number of zoster patients present with postherpetic neuralgia, characterized by severe pain that may persist for long periods after the rash has resolved. Some rare complications of zoster include encephalitis, conjunctivitis, keratitis and other eye disorders, Ramsey-Hunt syndrome, neurogenic bladder, and transverse myelitis.
Epstein-Barr virus (EBV; also known as human herpesvirus 4 [HHV-4]) and cytomegalovirus (CMV; also known as HHV-5) logically belong in the same chapter. Both of these human herpesviruses are major pathogens for the immunocompromised host, and both are capable of causing disease during primary infection, reactivation, or superinfection. They, along with BK polyomavirus, are the feared viral triumvirate that complicates hematopoietic cell transplantation (HCT) or solid organ transplantation. This chapter emphasizes recognition of EBV and CMV diseases and focuses on the clinical relevance of laboratory findings.
Herpesviruses 6, 7, and 8 are the most recently described members of the human herpesvirus family. Like other herpesviruses, they have the ability to establish a latent or persistent infection following primary infection, and reactivation may occur in healthy and immunocompromised people in response to different stimuli. A variety of methods are available or under development for the laboratory diagnosis of each virus, including viral isolation in cell culture, demonstration of viral antigens or nucleic acids in body fluids or tissues, and serology for detection of virus-specific antibodies. This chapter focuses on the immunologic and molecular diagnosis and monitoring of infections with human herpesvirus 6 (HHV-6), HHV-7, and HHV-8, and provides information on the unique features of the epidemiology and biological and clinical characteristics of these viruses.
Autonomous parvoviruses capable of helper-virus-independent replication have been isolated from many animal species. The human serum parvovirus B19 was accidentally discovered in 1975 in healthy donor blood used in the development of hepatitis B virus surface antigen diagnostic tests. To date, three B19-type genotypes have been described: types 1 (B19), 2 (A6/K71), and 3 (V9). Disease variation has not been reported amongst the three genotypes (1–3). Bocavirus, another parvovirus, has been associated with pulmonary infection (4). PARV4 has been identified in a parenteral drug abuser, but human disease causation has not been confirmed (5, 6). Recently, a proposal was submitted to the International Committee on Taxonomy of Viruses to reclassify B19 as primate erythroparvovirus type 1. The most frequent clinical presentation of B19 infection is erythema infectiosum, or fifth disease, a common childhood exanthem. Application of sensitive molecular biological and immunological methods to viral diagnosis has allowed recognition of the ever-expanding spectrum of clinical presentation (7).
Respiratory viruses are among the most common acute viral infections affecting humans and contribute appreciably to school and to work absenteeism. Globally, acute respiratory tract infections (ARTI) cause frequent morbidity and mortality among children under the age of 5 years, and are a major health care burden (1). Among the very young and the very old, as well as those with chronic medical conditions, respiratory viruses contribute substantially to medical visits, prescription of antibiotics and antivirals, hospitalizations, and deaths.
Measles virus, also called rubeola, and mumps virus are both RNA viruses of the family Paramyxoviridae, subfamily Paramyxovirinae; measles is in the Morbillivirus genus, and mumps virus is in the Rubulavirus genus. Rubella virus is also an RNA virus but is a member of the Togaviridae family. These three viruses, despite differences in their families and genera, are often considered together because the epidemiology of the infections they produce in humans and the preventive measures used against them are similar for all three. Measles and rubella, both of which produce rash-associated illness, and mumps, which typically infects the parotid glands, were all included among the expected illnesses of childhood in the United States prior to the introduction of vaccination programs in the late 1960s. Measles epidemics involving 500,000 to 700,000 cases occurred every 2 years (1), approximately 180,000 cases of mumps were reported annually (2), and approximately 58,000 rubella cases that included 60 congenital rubella syndrome (CRS) cases (3) were seen annually.
The term “hepatitis” refers to the inflammation of the liver, and in this chapter the five so-called major or primary viruses that cause this inflammation are described. These are the viruses hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis delta virus (HDV), and hepatitis E virus (HEV). In Table 1, the characteristics of these hepatitis viruses are described. Although there are also secondary viruses that can infect the liver and cause hepatitis, such as cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1, and enterovirus, the five primary viruses account for at least 95% of hepatitis virus infections. They primarily cause an infection of the liver and may infect other organs, while the secondary viruses infect the liver only during the course of a systemic infection.
The healthy human enteric tract is populated by a complex community of microbiota in synergistic balance with its host. Infection with a pathogenic virus can cause intestinal damage, inflammation, and diarrhea that alter the gut environment. The ability to diagnose pathogenic viruses rapidly and accurately is important in providing appropriate patient care and in controlling virus spread.
The arthropod-borne viruses (arboviruses) represent a diverse amalgam of more than 500 animal viruses that are grouped together because of their ability to replicate in both vertebrates and arthropods. While more than 150 arboviruses are known to cause human illness or infection, the list of the most medically important viruses is considerably shorter (Table 1) (1–3). Because of the vast number of agents that potentially should be considered in a differential diagnosis, the use of epidemiologic, ecologic, and clinical data to guide the choice and interpretation of clinical laboratory diagnostic tests is essential. Most arboviruses are transmitted seasonally in specific geographic locations or ecological habitats. The patient's history of travel, activities, and potential exposures to arthropods or habitats associated with arbovirus transmission provides vital data for selection of appropriate antigens and relevant diagnostic approaches. Knowledge of the patient's immunizations against yellow fever (YF), Japanese encephalitis (JE), or tick-borne encephalitis (TBE) viruses is also important for the proper interpretation of serological results. Although the laboratory diagnosis of arboviral infections still relies chiefly on serology, other approaches that directly detect viral antigen or genomic material and not antibodies are now routine.
Hantavirus is a genus in the family Bunyaviridae that includes small mammal-borne viruses. These viruses are causative of severe human diseases such as hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe and cardiopulmonary syndrome (HCPS) in the Americas. Hantaviruses are 80- to 120-nm enveloped RNA viruses. Their negative-sense single-stranded genome has approximately 12,000 nucleotides and is divided into three fragments named S (small), M (medium), and L (large) (1). The S fragment encodes the nucleoprotein (N), the M segment encodes the envelope glycoproteins (Gn and Gc), and the viral RNA (vRNA)-dependent RNA polymerase (RdRp), the largest viral protein (250 to 280 kDa), is codified by the L segment. The viral RNA segments are circular, include base-paired inverted complementary sequences at the 3′ and 5′ ends, and wrap up by the N protein (1). An open reading frame encoding NSs can be found in American hantavirus and in the vole-borne Puumala and Tula viruses, all evolutionarily related (2, 3).
Rabies virus (RABV) and RABV-related lyssaviruses are the causative agents of zoonotic viral encephalitis, with a case/fatality ratio approaching 1:1. These viruses are endemic in a number of terrestrial mammals and bat species throughout much of the world. Lyssaviruses (family Rhabdovirus, order Mononegavirales, of which RABV is the type species) are characteristically bullet-shaped particles averaging 75 by 180 nm with a single-stranded, negative-sense RNA genome encoding five proteins. In genome order, these proteins are nucleoprotein (N), which tightly encapsulates the genome; phosphoprotein (P), which was formerly referred to as the nonstructural (NS) protein; matrix (M) protein; glycoprotein (G), the primary target of neutralizing antibodies, which is found spread over the surface of the virus; and polymerase (L), the RNA-dependent RNA polymerase. Genetically and antigenically more diverse in glycoprotein than in nucleoprotein, the different lyssavirus variants are each associated with a particular host species. The major reservoir of RABV in Asia is the dog, historically and currently the cause of most human rabies cases; wildlife, including foxes, coyotes, skunks, raccoons, and bats, carry RABV or RABV-related viruses in diverse geographic regions. Effective vaccines and postexposure prophylaxis (PEP) regimens consisting of active and passive vaccination have been available for some time, and where animal reservoirs of the virus have been controlled by vaccination and PEP is readily available, human rabies is no longer the well-known disease of antiquity. However, in Asia, where dog rabies persists, human rabies is not uncommon, with cases estimated to occur at a rate of over 50,000 a year. Elsewhere, human rabies is sporadic, generally resulting from the bite of an infected dog while a traveler is in a region where rabies is endemic or from an interaction with an infected animal when the possibility of virus transmission is not recognized. As RABV can infect all mammals, the virus may be transmitted between a reservoir species and humans by intermediate species that are not normally associated with rabies, such as cats. However, the more common cause of unrecognized infection is through contact with an infected bat. In such cases, the bite or scratch responsible for the infection may be so minor as to go unnoticed.
Human T-cell lymphotropic virus type 1 (HTLV-1) was the first pathogenic human retrovirus to be discovered. It was isolated in 1979 from the peripheral blood lymphocytes of a patient with adult T-cell leukemia/lymphoma (ATLL) (1). Following years of research to find a retroviral agent involved in ATLL, Yoshida and Yamamato isolated a retrovirus from an ATLL patient in 1986 that they named adult T-cell leukemia virus. Soon, the similarities between HTLV-1 and adult T-cell leukemia virus were confirmed, and the name HTLV-1 was chosen. Shortly thereafter, the closely related virus HTLV-2 was isolated from the splenic cells of an individual with hairy cell T-cell leukemia (2).
Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a large group of transmissible, progressive, and invariably fatal neurodegenerative conditions that affect both animals and humans (1–5). Prion diseases are unique in that they can be inherited, occur sporadically, or can be acquired by infection (1, 3–5). As described below, the infectious agent in the prion disease is composed mainly or entirely of an abnormal conformation of a host-encoded glycoprotein called the cellular prion protein (PrPC). The replication of prions involves the recruitment of the normally expressed prion protein (PrPC) structure, which is largely alpha-helical, into a disease-specific conformation (PrPSc) that is rich in beta-sheets and that can adopt a fibrillar aggregated structure that is characteristic of many of the deposits found in the brains of TSE-affected species. In contrast to the protease-sensitive PrPC, the beta-sheet conformation along with the aggregation properties of PrPSc makes this protein partially resistant to proteolytic digestion (6). Furthermore, this posttranslational modification of PrPC into the abnormal, infection-associated isoform, PrPSc, is believed to be the principal molecular basis underlying prion diseases. Animal prion diseases include scrapie of sheep and goats, bovine spongiform encephalopathy (BSE) or mad cow disease, chronic wasting disease (CWD) of cervids (predominantly mule deer and elk), transmissible mink encephalopathy (TME), feline spongiform encephalopathy, exotic ungulate spongiform encephalopathy, and spongiform encephalopathy of nonhuman primates. Although some cases of sporadic atypical scrapie and BSE have also been reported, most animal prion diseases occur via the acquisition of infection from contaminated feed or via exposure to environmental contaminants. Scrapie and CWD are naturally sustaining epidemics. The human prion diseases can be sporadic, inherited, or acquired. Sporadic human prion diseases include Creutzfeldt-Jakob disease (sCJD), fatal insomnia, and variably protease-sensitive prionopathy (VPSPr) (3, 4). Genetic prion diseases are caused by inheritance of autosomal dominant mutations in the host PRNP gene, which encodes the normal cellular PrPC and includes genetic CJD (gCJD), fatal familial insomnia (FFI), and Gerstmann-Sträussler-Scheinker syndrome (GSS) (3, 4). Acquired human prion diseases account for only 5% of cases of human prion disease. They include kuru, iatrogenic CJD (iCJD), and variant CJD (vCJD) (3, 4), which was transmitted to humans from affected cattle via meat consumption. The transmission of BSE to humans has resulted in more than 200 cases of vCJD and has raised serious public health concerns. All prion diseases have long incubation periods but are typically rapidly progressive once clinical symptoms begin. Currently, there are no effective treatments for prion diseases, although increased understanding of their pathogenesis has recently led to the promise of effective therapeutic interventions. Numerous therapeutic approaches are under development both for the prevention of prion disease prior to or shortly after exposure and for treatment of already symptomatic disease (2, 7–10).
The human immunodeficiency virus (HIV) is the etiologic agent of AIDS. The clinical manifestations of AIDS were first recognized in 1981 (1). Search for the cause of this severe cellular immune dysfunction led to isolation of lymphadenopathy-associated virus in 1983 (2). The following year, additional researchers isolated cytopathic retroviruses from persons with AIDS, which they termed human T-lymphotrophic virus type III (3, 4). These viruses were soon confirmed to be identical, and in 1986 the International Committee on Taxonomy of Viruses named the virus causative of AIDS HIV (5). Genetic sequences of HIV have been identified retrospectively in human plasma specimens from as early as 1959 (6). It is estimated that approximately 75 million people worldwide have become infected with HIV. In the United States, the Centers for Disease Control and Prevention (CDC) estimates that 1.1 million persons are living with HIV.
Patients with primary immunodeficiency diseases most often are recognized because of their increased susceptibility to infection (chronic or recurrent infections without other explanation, infection with an organism of low virulence, or infection of unusual severity). However, these patients may also present with autoimmune or inflammatory disorders (e.g., hemolytic anemia, inflammatory bowel disease, vasculitis, or systemic lupus erythematosus) or as part of a syndrome complex (Table 1). Finally, in the future we will see an increasing number of people who are identified as having an immunodeficiency because of an abnormal newborn-screening test for T-cell receptor-excision circles (TRECs) or a mutation identified by genome sequencing.
The history of newborn screening (NBS) in the United States began in 1963, when Guthrie demonstrated that phenylketonuria (PKU) can be detected using dried blood spots (DBSs) on filter paper from newborns (1). Neonatal testing for PKU proved to be inexpensive, sensitive, specific, and amenable to high-throughput screening. The ability to screen neonates for other serious diseases increased dramatically, and in 1968, Wilson and Jungner published a landmark report that included guidelines for population-based health screening (Table 1) (2). This report tried to balance the desire for early detection and treatment of disease with the potential harms to patients, family, and society. The report defined principles for NBS programs and has undergone revisions to address concerns for both newborn screening and population-based screening programs in general (Table 1) (3–5).
This chapter is dedicated to describing combined immunodeficiencies (CIDs) and useful diagnostic assays. Here, a CID is defined as a defect that affects the two most prominent arms of the adaptive immune system: T cells and B cells. The molecular defect may directly affect both cell types or just one cell type, specifically T lymphocytes, with indirect effects on B-cell function. CIDs are distinguished from severe combined immunodeficiencies (SCIDs) because T-cell numbers and function are higher than per established criteria for SCID and thus may not be detected on SCID newborn screening (NBS). Several CIDs are attributed to distinct genetic mutations in SCID-associated genes. In recent years, the field of immunology has witnessed increased reporting of unique CIDs affecting isolated families or small numbers of individuals. This increase is attributable to the advent of improved genetic testing, such as whole-exome sequencing. In Table 1, we provide a comprehensive list of CIDs, their immunophenotypes, and useful diagnostic tests. Common themes for CIDs include aberrant T-cell development, function, cytoskeletal regulation, and survival. These defects translate to clinical problems secondary to immune deficiency and, in many instances, immune dysregulation. This chapter does not include in-depth description of all CIDs but rather focuses on categorical descriptions and highlights more prevalent genetic abnormalities along with overviews of diagnostic assays for CID confirmation.
Antibody deficiency can be defined as a condition characterized by a reduction in serum immunoglobulin concentrations below the fifth centile for age. Antibody deficiency may affect all classes of immunoglobulins or may be confined to a single isotype.
Complement deficiencies comprise a small but important category of primary immune deficiency diseases. Complement deficiency states can also be acquired from in vivo activation or inhibition of the complement system, temporarily depleting components faster than they can be replaced or causing changes in control of the system. Complement plays an important role in the host's response to infection by directly killing bacteria, neutralizing viruses, or coating microbial surfaces with complement fragments that enhance uptake and killing by phagocytes, a process called opsonization that ensures that objects identified as foreign by complement are dealt with quickly and efficiently. Complement serves as a link between many of the activities of acquired immunity, with ties to diverse cell signal responses and other defense mechanisms, as an ever-increasing number of related conditions are identified (1).
Concern about the neutrophil status of a patient is usually raised on the basis of the frequency, severity, and distribution of a specific infectious agent(s) involved in one or more episodes that are, or are thought to be, infectious. The clinical presentations of patients with neutrophil disorders usually share a few common features: gingivitis, periodontal disease, and oral ulceration. Cutaneous infections with Staphylococcus aureus are often recurrent and can be severe. In neutrophil disorders characterized by inadequate inflammation (neutropenia, leukocyte adhesion deficiency [LAD], Chédiak-Higashi syndrome, and specific granule deficiency), infections can extend locally and subcutaneously with little reaction until marked destruction has taken place. Clinically relevant neutrophil abnormalities fall into several broad categories: neutropenia, abnormalities of neutrophil adherence and locomotion, abnormalities of neutrophil granule formation or content, and abnormalities of killing. With the widespread use of therapies which modulate the immune system either by design (e.g., steroids and monoclonal antibodies) or incidentally (e.g., cytotoxic chemotherapy), the most common causes of immunodeficiency are iatrogenic. The recognition, characterization, identification, and cloning of disease-related genes and, in some cases, genetic correction of immune defects are progressing rapidly. As specific and genetic treatments become available, making the correct diagnosis early takes on greater therapeutic importance (1).
Genetic defects in natural killer (NK) cell and cytotoxic T lymphocyte (CTL) function generally lead to one of two outcomes: (i) life-threatening and/or severe chronic infections with viruses, particularly from the Herpesviridae family member viruses Epstein-Barr virus, cytomegalovirus, herpes simplex viruses, and varicella-zoster virus but also other viruses such as human papillomaviruses; or (ii) a life-threatening hyperinflammatory disorder called hemophagocytic lymphohistiocytosis (HLH).
The prevalence of allergic diseases has risen dramatically throughout the world in recent years, creating a major health care burden. While immunoglobulin E (IgE) has been recognized to play a central role in the pathogenesis of human allergic conditions since 1967, a multitude of inflammatory mediators and cells are now known to contribute to these common phenotypes. Section L covers laboratory investigations useful in the diagnosis and management of allergic disorders. Important topics covered include the allergen, in vivo and in vitro assays to screen for the presence of allergen-specific IgE and other mediators of allergic inflammation, and analytic approaches to diagnosing food allergy, hypereosinophilic syndromes, and mast cell disorders.
Allergic reactions and allergic diseases are the most common human disorders of immune regulation. Diseases may include localized responses in the skin and various portions of the airway, or systemic responses characterized by extensive skin involvement, severe airway compromise, or cardiovascular collapse. Mechanisms include mast cell or basophil activation by the cross-linking of allergen-specific homocytotropic IgE, cellular infiltration following mast cell or basophil mediator release, complement activation, the deposition of immune complexes in susceptible tissues, or the infiltration of activated T-lymphocytes. The degree of impairment from allergic disease varies widely, with most reactions posing minor inconvenience, but with rare episodes requiring intensive—and sometimes unsuccessful—interventions to prevent death.
Nearly 50 years have passed since IgE was identified as the reagin or serum antibody that sensitizes skin mast cells and circulating basophils and mediates immediate-type hypersensitivity reactions in humans (1, 2). Since IgE's identification, the clinical immunology laboratory has provided the clinician with analytical measurements that aid in the diagnosis, management, and research of the natural history and epidemiology of IgE-mediated diseases. Total and allergen-specific IgE antibody are the primary analytes measured clinically to support the diagnosis of human allergic disease. Antigen-specific IgG antibodies are measured as a research analyte to identify chronic allergen exposure and to monitor potential blocking antibody activity following immunotherapy.
Assays facilitating the measurement of mediators and markers of allergic inflammation have evolved during the past 40 years, arising from two primary discoveries: first, that immunoglobulin E (IgE) is the source of reaginic activity in serum, and second, that tissue mast cells and blood basophils—cells expressing the high-affinity receptor for IgE (FcεR1)—respond to allergen by releasing the most relevant mediators underlying immediate hypersensitivity reactions. Naturally, skin testing has long been used to assess the in vivo mast cell response to allergen. Basophils, however, by virtue of their accessibility and the fact that they are the sole source of many of these mediators among blood leukocytes, continue to dominate the performance of in vitro assays assessing IgE-mediated responses.
Food allergy is defined as an adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food protein, and is distinguished from food intolerance (e.g. lactose intolerance), which is typically nonimmunologic in origin (1). Food allergic disorders can be divided into those that are immunoglobulin E (IgE)-mediated and those that are non-IgE mediated. Disorders with acute onset of symptoms, defined typically as occurring within 2 hours after ingestion, are usually mediated by IgE antibodies, while subacute and chronic food allergic disorders may be cell mediated (primarily T cell) or of mixed origin with both cell-mediated and IgE-associated mechanisms. The subacute and chronic food allergic disorders primarily affect the gastrointestinal tract. Table 1 lists examples of IgE-, cell-, and mixed IgE- and cell-mediated disorders.
Whereas mild to moderate eosinophilia has been reported in as many as 0.1% of North American outpatients (1) and may be due to seasonal allergies, asthma, or other common conditions, marked eosinophilia (>1,500/mm3) is relatively infrequent and should always prompt a diagnostic evaluation. The differential diagnosis of marked eosinophilia is broad and includes secondary causes of eosinophilia (Table 1), such as hypersensitivity reactions, helminth infection, and neoplastic and inflammatory disorders, as well as several disorders for which eosinophilia is thought to be the primary etiology. In many cases, a thorough diagnostic evaluation will reveal a secondary cause of the eosinophilia, and appropriate treatment can be instituted. In other instances, a well-defined, single-organ-restricted, primary eosinophil disorder, such as eosinophilic esophagitis, eosinophilic fasciitis, or eosinophilia cystitis, is identified. Once secondary causes and alternative diagnoses have been excluded, however, a systemic primary eosinophil disorder should be considered.
Systemic autoimmune diseases are often associated with the production of autoantibodies that recognize a diverse array of cytoplasmic and nuclear antigens. These autoantibodies are used as adjuncts in the diagnosis of autoimmune disease, for monitoring disease activity and severity, and for predicting the outcome of autoimmune disease. As the diagnosis of systemic autoimmune disease is not always straightforward, autoantibody testing has the potential to improve our ability to diagnose complex autoimmune disorders. However, autoantibody test results, particularly those based on enzyme-linked immunosorbent assays (ELISAs) and other solid-phase assays, must be interpreted with caution due to their high sensitivity and the possibility of false-positive results. It is important to interpret the results of autoantibody tests in light of both the clinical context and the methodology employed. In some cases, confirmatory testing, analogous to the use of Western blotting to verify positive ELISA test results for HIV infection, is warranted.
Autoantibodies are historical hallmarks in the establishment of the concept of autoimmunity and in the definition of the clinical limits of several autoimmune diseases. On a day-to-day basis, autoantibodies are helpful elements not only in the diagnosis of autoimmune diseases, but also frequently in the establishment of prognosis and in the monitoring of disease activity.
Autoantibodies directed against intracellular antigens are characteristic features of a number of human autoimmune diseases and certain malignancies (1–3). Studies of systemic autoimmune rheumatic diseases have provided strong evidence that autoantibodies are maintained by antigen-driven responses (4, 5) and that autoantibodies can be reporters from the immune system, revealing the identities of antigens involved in disease pathogenesis. Historically, autoantibody detection and analysis have relied on a number of different technologies, such as hemagglutination and particle aggregation, immunodiffusion, indirect immunofluorescence (IIF), complement fixation, counterimmunoelectrophoresis (CIE), Western and dot blotting, immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA), and on functional assays that demonstrate inhibition of the catalytic or other functional activity of the antigen of interest. These technologies have limitations because they tend to be labor-intensive and time-consuming, are limited in throughput, are semiquantitative, and are not adaptable to leading-edge research. Immunodiffusion has been used for over 50 years, and it is still used in some clinical laboratories because it is inexpensive and has high specificity, but it lacks sensitivity and can take up to 48 h before precipitin lines are interpretable. Western blotting is more costly and time-consuming, and not all autoantibodies are detected by this technique. For example, in the SS-A/Ro system, it has been observed that IP techniques are required to identify some sera that contain antibodies reacting only with the “native” SS-A/Ro particle (6). IP protocols that use extracts from [35S]methionine-labeled cells are not suitable for the detection of all autoantibodies, such as antibodies to Ro52/TRIM21 protein (7). ELISA techniques have rapidly advanced, but highly specific, sensitive, and reliable assays that use highly purified or recombinant proteins are limited by intermanufacturer and interlaboratory variation of results (8). Immunodiffusion and CIE generally favor high-titer sera and often cannot discriminate multiple autoantibody responses that are characteristic of systemic autoimmune rheumatic disease sera.
This chapter deals with immunological tests that are useful in the diagnosis and monitoring of systemic lupus erythematosus (SLE). We review the detection and quantification of antibodies against self-antigens (autoantibodies), measurement of complement levels, and the interferon gene expression signature.
Certain autoantibodies can be clinically useful biomarkers associated with a particular disease and/or clinical features. Some of them, called disease marker antibodies, are highly specific for a particular diagnosis and have predictive value for the development of the disease, and they are included in classification criteria for systemic autoimmune rheumatic diseases (1, 2). The majority of disease-associated autoantibodies in systemic lupus erythematosus and scleroderma (systemic sclerosis) have been known for decades. However, autoantibody research in polymyositis/dermatomyositis (PM/DM) has been highly active in recent years as several new, clinically important autoantibody specificities with strong clinical impact have been identified, such as antibodies to transcription intermediary factor 1γ/α (TIF1γ/α; p155/140), which are frequently found in cancer-associated DM (3–5); and to melanoma differentiation-associated gene 5 (MDA5), associated with clinically amyopathic DM (CADM) with rapidly progressive interstitial lung disease (ILD) (6–9). Autoantibodies that are found in PM/DM are often classified into myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (10, 11). MSAs are found almost exclusively in PM/DM among systemic rheumatic diseases, although some are also found in patients classified into idiopathic ILD, such as anti-PL-12 and anti-KS antibodies (12, 13).
Systemic sclerosis (SSc) or scleroderma is a connective tissue disease characterized by excessive fibrosis, microangiopathy, and the presence of circulating autoantibodies to various cellular components (1). Clinical presentation is highly heterogeneous in patients with SSc: some have only Raynaud's phenomenon and sclerodactyly without any symptomatic organ involvement for >10 years, but others have progressive functional impairment in lungs, heart, kidneys, or gastrointestinal tract, leading to death. Distinct specificities of antinuclear antibodies (ANAs) are selectively detected in SSc patients and are associated with unique disease manifestations (2). The detection of individual SSc-related ANAs is useful in the diagnosis, disease subgrouping, and prediction of future organ involvement and prognosis. Therefore, SSc-related ANAs are important biomarkers in routine rheumatology practice.
Rheumatoid arthritis (RA) is a chronic systemic disease characterized by autoimmune-mediated joint destruction. RA affects 1% of the global population with a predilection for females. The pathogenesis of RA has not been fully elucidated; however, it is thought to be due to an environmental trigger stimulating an immune response in a genetically susceptible host. The resultant inflammatory cascade mediates synovial proliferation and ultimately joint destruction. Disease expression is not limited to the joints. Extra-articular manifestations occur in the lungs, eyes, skin, and the cardiovascular system culminating in significant morbidity and premature mortality (1). Moreover, numerous studies show substantial irreversible joint damage occurs within the first 2 years of disease onset. Thus, early diagnosis and aggressive treatment is paramount in preventing morbidity and mortality (2).
Antiphospholipid antibody syndrome (APS) is an acquired thrombotic disorder caused by a heterogenous group of antibodies directed against plasma proteins that bind phospholipids. These are termed “antiphospholipid antibodies” (aPLs), and while they are required for the diagnosis for APS, they can also be detected in otherwise healthy individuals. The clinical manifestations of APS can be divided into thrombotic and obstetrical. Thrombotic complications include both venous and arterial thrombosis. Obstetrical complications include recurrent first trimester miscarriage and second trimester fetal demise, as well as other pregnancy complications like preeclampsia and placental insufficiency (1). APS is classified as “primary” when not associated with an underlying disease and accounts for over 50% of cases (2). Secondary APS is the distinction given to APS associated with an underlying autoimmune connective tissue disorder such as SLE.
Inflammation of and damage to blood vessel walls are the shared defining features of all vasculitides. The symptoms vary depending on the size of the vessels affected, the organs they serve, the underlying cause of the vasculitis, and the activity of the disease. The vasculitic diseases are rare. Since there are only 20 to 50 new cases of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitides per 1,000,000 people per year (1, 2), a systematic and thorough diagnostic algorithm is needed to correctly identify patients with the disease (3, 4). Missed or delayed diagnosis can result in permanent organ injury or even death (5, 6).
IgG4-related disease (IgG4-RD) is a multiorgan immune-mediated condition that mimics many malignant, infectious, and inflammatory disorders (1–3). The diagnosis links numerous conditions once regarded as isolated, single-organ diseases that existed outside of any known underlying systemic condition (Table 1). The histopathological findings in IgG4-RD are consistent across the wide range of organ systems that can be involved. IgG4-RD is often confused with a malignancy upon presentation because it has a tendency to form mass lesions. In addition, IgG4-RD can mimic a host of immune-mediated conditions, e.g., primary sclerosing cholangitis, idiopathic interstitial lung disease, Sjögren's syndrome, granulomatosis with polyangiitis, and idiopathic membranous nephropathy.
Immunologic biomarkers are intrinsic to the field of rheumatology both for making a clinical diagnosis and in potentially detecting early signs of disease in at-risk individuals when symptoms are absent. In this brief chapter, we discuss developing and future directions in the use of biomarkers in rheumatology with an emphasis on rheumatoid arthritis. We discuss the role of the “mechanistic” biomarker, a subtype of the biomarker that measures a process directly linked to disease pathogenesis, thus increasing both diagnostic reliability and potentially identifying phenotype-targeted therapies. As such mechanistic biomarkers have begun a revolution in the approach to cancer, we attempt to provide parallels as to how such markers can be applied to diagnosis and targeted treatment in the rheumatic diseases.
Autoimmunity is common, while autoimmune disease is not. It is not clear how or why a benign autoimmune response becomes “malignant,” thereby leading to pathogenic changes of a target organ. Few autoimmune diseases fulfill the strict criteria by which the autoimmune response is directly related to pathogenesis. However, many findings, either cellular or humoral, can provide strong circumstantial evidence that autoimmunity is strongly associated with a particular disease. Often, the humoral responses detected in the laboratory are best considered to be markers rather than causes of disease. The autoantibodies are useful for diagnosis or prognosis, for excluding other conditions, for classifying disease, or for monitoring therapy. Whenever we test for autoimmune disease by evaluating autoantibodies, we must consider that individuals without evidence of disease may also exhibit these autoantibodies. Therefore, all results must be evaluated in context with the entire clinical picture; autoantibodies are rarely the sole criterion for disease. However, since autoantibodies are a significant marker of disease, often developing years before symptoms are present, the autoantibodies may indicate future disease.
Chronic lymphocytic thyroiditis (CLT), also known as Hashimoto thyroiditis, is an autoimmune disease characterized by lymphocytic infiltration of the thyroid gland, with the concomitant production of autoantibodies to thyroid antigens, primarily thyroglobulin and/or thyroperoxidase (TPO), formerly known as microsomal antigen (1). Although few epidemiological data are available, the prevalence of CLT is estimated to be 1 in 1,000 people, with an incidence of 0.2 to 2% and a female-to-male ratio of about 18:1 (1). Clinical signs and symptoms manifest slowly and may involve many systems of the body (1). Accumulations of hydrophilic mucoproteins with edema, a condition called myxedema, affects skin and connective tissue and can affect the appearance of the individual. Lethargy may ensue, with a loss of mental acuity. Systems commonly affected are the gastrointestinal tract, the hemopoietic system, the endocrine system, and the urogenital system (1). Enlargement of the thyroid gland due to lymphocyte invasion, called goiter, is a frequent manifestation, although there is an atrophic variation. Demonstration of autoantibodies to the thyroid antigens aids in the diagnosis of CLT, distinguishing it from other causes of hypothyroidism.
Myasthenia gravis (MG) is a disease of striated muscles which clinically manifests as weakness. It is caused by impaired neuromuscular transmission due to a reduction in the number of receptors for the neurotransmitter acetylcholine (ACh) at the postsynaptic myoneural junction. This reduction is caused predominantly by the action of anti-acetylcholine receptor (anti-AChR) antibodies in most instances. The disease occurs with a reported prevalence of 0.5 to 5/100,000 and an incidence of 0.4/100,000/year. MG can occur at any age; however, it typically presents in the second and third decades of life, with a later peak occurring after age 50 (late-onset disease). A female preponderance (3:1 to 4:1) has been reported in the first 40 years of life; thereafter, the incidences are comparable between the sexes.
Peripheral neuropathies constitute a diverse group of diseases caused by a wide range of genetic, toxic, metabolic, and inflammatory insults to the peripheral nervous system. A considerable proportion of neuropathies are believed to have an autoimmune basis, either as a feature of systemic autoimmune diseases, vasculitides, or paraneoplastic or postinfectious syndromes, in association with lymphoproliferative diseases, or as isolated peripheral nerve syndromes. In clinical practice, the cause of sporadic neuropathies is often obscure, resulting in the frequent use of multiple screening tests to aid in diagnosis. Over the last 20 years, there has been a widespread increase in the use of antiganglioside antibody assays as diagnostic tools, based on the recognition from research studies that gangliosides are important autoantigens in many patients with autoimmune peripheral nerve disorders (1).
The immunodetection of autoantibodies in autoimmune liver disease has been technically difficult because of the following: (i) serum autoantibodies from these patients usually react to a broad spectrum of antigens; (ii) some of these autoantibodies may have low titers; (iii) the biochemical nature of these autoantigens is unknown; and (iv) many autoantigens have low concentrations, and their biochemical purification often requires sophisticated procedures. The development and application of immunohistochemical, biochemical, and molecular biological techniques have provided new approaches to the study of autoimmune diseases and, in particular, the immunological detection of autoantigens. This is exemplified by two autoimmune diseases of the liver, namely, primary biliary cholangitis (PBC, formerly known as primary biliary cirrhosis) and autoimmune hepatitis (AIH). This chapter focuses on the detection of antimitochondrial autoantibodies (AMA) in PBC and the detection of liver kidney microsomal (LKM) antibodies in AIH.
Acute myocardial injury (AMI) most often results from a lack of sufficient blood supply to the myocardium. When patients present with symptoms of chest pain, rapid diagnosis of potential AMI and characterization of the extent of cardiac injury are essential for delivering appropriate treatment, e.g., intervention with thrombolytic therapy or angioplasty. Such interventions are aimed at minimizing the risk of (further) cardiac injury and death. Distinguishing patients with unstable angina from those with AMI is necessary to triage whether patients may be managed in the outpatient setting versus inpatient admission and immediate intervention. Serologic measurements of soluble biomarkers released during myocyte necrosis are used in conjunction with electrocardiograms and with more sophisticated imaging techniques like echocardiography and perfusion scintigraphy. Serum biomarkers have the greater advantage of detecting recent myocardial injury, which is not easily distinguishable by cardiac imaging techniques in patients with clinical evidence of preexisting heart disease (Table 1).
Celiac disease (CeD) and inflammatory bowel disease (IBD) are chronic inflammatory diseases affecting the gastrointestinal tract. They share certain characteristics, including autoimmune features, the presence of both gastrointestinal and systemic clinical symptoms, and pathogenic contributions by genetic and environmental factors. However, there are several stark differences that must be appreciated. The immunogenic mechanisms of the two diseases are unique, with differing contributions by the innate and adaptive immune responses. Histologically, CeD is distinct from IBD, which is useful diagnostically as well as for gaining insight into disease mechanisms. Lastly, the role of diagnostic laboratory testing, both serologic and genetic, differs greatly between CeD and IBD. The purpose of this chapter is to provide a summary of what is currently known about the pathogenesis, epidemiology, clinical presentation, and diagnostic testing for CeD and IBD.
Immune-mediated hemolytic anemia can be broadly divided into two categories: alloimmune and autoimmune (1–4). Autoimmune hemolytic anemia may be further classified according to the immunoglobulin type of the autoantibody and its optimum temperature of reactivity (1–4). Warm autoimmune hemolytic anemia is due to IgG autoantibodies that bind to erythrocytes optimally at 37°C, while cold autoimmune hemolytic anemia is caused by IgM autoantibodies that preferentially react with red cells at low temperatures. A mixed-type autoimmune hemolytic anemia may occur when both warm IgG and cold IgM autoantibodies are implicated in clinical hemolysis. The rarest form of autoimmune hemolytic anemia, paroxysmal cold hemoglobinuria (PCH), is associated with IgG autoantibodies that are biphasic, sensitizing erythrocytes at lower temperatures and activating complement as the temperature increases to 37°C.
Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by a platelet count of <100,000/μl in the absence of any underlying cause. The incidence of newly diagnosed ITP in adults ranges from 1.6 to 3.9 per 100,000 person-years. The female-to-male ratio ranges from 1.2 to 1.9. The general consensus is that the diagnosis is one of exclusion based on patient clinical history, physical examination, review of medication history, and performance of a complete blood count with review of the peripheral blood smear. One can classify the illness based on duration: (i) newly diagnosed with a duration of up to 3 months, (ii) persistent ITP of 3 months to 1 year, or (iii) chronic ITP of >1 year (1).
The identification of immune reactivity against self components is a key element in the characterization of autoimmunity and autoimmune diseases. This autoimmune reactivity is most frequently monitored by the detection of specific autoantibodies or the presence of T-cell reactivity to self peptides. Numerous studies have now demonstrated that distinct profiles of autoantibodies are seen in clinically distinct autoimmune syndromes, such as systemic lupus erythematosus, myositis, and insulin-dependent diabetes mellitus. Moreover, the autoantigens are targets of an adaptive immune response.
The National Cancer Institute has defined cancer as a group of diseases in which abnormal cells divide without control and are able to invade other tissues. Cancers can be broadly categorized into five groups depending on their origin: carcinoma (skin), sarcoma (bone, cartilage, fact muscle, and blood vessels), leukemia (blood-forming tissue and bone marrow), lymphoma/myeloma (cells of the immune system), and central nervous system (brain and spinal cord).
Tumor markers are molecules derived from patient body fluids or tissues that are measured and can provide information useful for the management of cancer patients. Applications include detection of cancer, monitoring of disease progression, and evaluation of the effectiveness of therapeutic regimens, among others. Henry Bence-Jones discovered the first tumor marker in 1846—a protein seen in acidified urine of patients with multiple myeloma (1). Since that time, many more tumor markers have been described for a variety of other cancers. The Food and Drug Administration (FDA) has approved several of these markers (Table 1), and many more are being investigated and evaluated for clinical use. The goal of this chapter is to review how tumor marker assays are evaluated, describe the applications of tumor markers, provide details on the detection of tumor markers using immunoassays, and then list examples of clinically useful tumor markers that are used in the clinical laboratory today. Although the last few years have witnessed a plethora of developments in molecular testing of tumor markers, these will not be discussed here, as we will restrict our discussion to immunoassay-based detection methods.
Malignancies of the immune system are primarily represented by the malignant lymphomas and a smaller number of nonlymphoid neoplasms that originate from cells involved in antigen presentation and processing. The diagnosis and classification of these tumors have benefited enormously from advances in cellular and molecular immunology, as well as from careful dissection of the molecular genetics of the cancers themselves. Our ability to correctly diagnose tumors of the immune system, which in many instances show little morphological variation, requires the careful application of both immunologic tumor markers as well as molecular (DNA- or RNA-based) tumor markers. Therefore, malignancies of the immune system are not only interesting models of normal immune cells but are also excellent examples of the variety of molecular and immunological approaches for assessing cancers.
Immunologic therapies are becoming increasingly widely utilized. The current interest in these therapies is due to considerable successes in treatment outcomes in a broad spectrum of human diseases targeted for biotherapy, including inheritable or acquired immunodeficiencies, autoimmune diseases, cancer, and persistent infections. Many new biologic agents are available for immune therapy, and antibodies, cytokines, activated or genetically modified immune cells, vaccines, and other immunomodulatory drugs are frequently used either as monotherapies or in combination with conventional therapies such as chemotherapy, radiation, surgery, and even behavior-modifying therapies. In cancer, biotherapies are expected to restore dysfunctional antitumor immune responses, activate immune cells, and help immune cells to reacquire normal homeostasis. In addition, tumors resistant to standard therapies are often sensitive to immune interventions, and this offers a promise of combinatorial therapies to overcome drug resistance.
Circulating tumor cells (CTC), first described in 1869 as cells in the blood identical to those of the cancer itself (1), originate from the primary tumor or metastatic deposits after intravasation through the tumor vasculature by mechanisms that are not completely understood (Fig. 1) (2). Although most CTC die in the circulation, a proportion of them have the ability to spread, seed, and proliferate in distant sites to form secondary metastases (3) (estimated to be 0.05%) or to reestablish in the organ of origin to form new tumors (4), as demonstrated in short-term proliferation experiments (3, 5). First described by Paget in 1889, in his “seed and soil” hypothesis (6), the process of metastatic dissemination is driven by chemokines and integrins specific to the tumor type and by the adhesive interactions of the CTC with the vasculature and extracellular matrix components (4, 7–9). As an example, β4 integrin signaling promotes tumor cell adhesion to the endothelium through ErbB2-mediated vascular endothelial growth factor secretion and has been involved in metastatic development (10). Additionally, CTC have the ability to secrete stem cell-like factors to promote proliferation and self-renewal (11, 12). Microembolic clusters of CTC, isolated by a variety of techniques, are associated with a particularly poor prognosis, which may be related to the enhanced activation of survival pathways by cellular contact and to mechanical capture in the microvasculature, further enabling metastasis formation (13, 14).
The scope of immunogenetics and histocompatibility testing in transplantation has changed dramatically over the past 50 years. In organ transplantation, outcomes continue to improve as a result of better immunogenetics testing, immune suppression, and patient management; however, acute rejection and chronic rejection remain the biggest obstacles for successful transplantation. Since “crossmatching” was first described by Patel and Terasaki (1), the goal to provide accurate immunological risk assessment for transplant recipients remains unchanged. Histocompatibility and immunogenetics laboratories utilize multiple diagnostic assays to evaluate HLA compatibility between donors and recipients. Furthermore, testing has expanded to include immunogenetics systems other than those using HLA, such as killer immunoglobulin-like receptors (KIR) (2, 3), major histocompatibility complex (MHC) class I chain-related gene A (MICA) (4), angiotensin type 1 receptor (AT1R) (5), and genes encoding cytokines. The application of new state-of-the-art diagnostic tests that inform on the immune status of the transplant recipient has transformed the role of the immunogenetics laboratory from providing tissue-typing and crossmatching services to assessing immunologic risk, donor selection, and presentation of strategies for desensitization and therapeutic intervention. In addition, immunological risk assessment is no longer limited to the pretransplant period but has become increasingly recognized as a noninvasive tool for assessing acute and chronic rejection (6–8).
The characterization and clinical assessment of the human leukocyte antigen (HLA) genes has undergone significant advances over the last 50 years. As serological methods have given way to more-advanced molecular methods, our understanding of the complexity and polymorphic nature of the HLA genes has been substantially improved. From its basis in serological and cellular testing in the 1960s (antibody and mixed lymphocyte culture) (1–5), through two-dimensional electrophoresis and restriction fragment length polymorphism analysis in the 1970s and '80s (5, 6), the development of PCR in the mid-1980s revolutionized our molecular understanding of the HLA genes. From PCR, methods utilizing sequence-specific oligonucleotide probes (SSOs) and sequence-specific primers (SSPs) provided the means for more directly evaluating the highly variable sequence motifs within the HLA genes (7–10). Subsequently, in the 1990s, Sanger sequence-based typing (SBT) significantly advanced tissue typing and transplantation genetics (11–14) by providing an unprecedented molecular view of HLA polymorphism in the context of exonic variation. Most recently, next-generation sequencing (NGS) appears to definitively address HLA typing complexity, as it provides entire HLA gene characterization and haploid sequence determination.
Antibodies of the IgG class can damage tissues in a variety of ways, including (i) directly through complement activation, (ii) indirectly through the deposition of immune complexes, and (iii) indirectly through the recruitment of cytotoxic or inflammation-inducing cells. Very high levels of antibody result in hyperacute rejection and graft failure, an outcome that can easily be avoided by the performance of a lymphocyte crossmatch test prior to transplantation. In contrast, clear elucidation of the relevance of donor-reactive antibodies of various strengths and specificities and of antibodies that arise after transplantation has been hampered by inadequate technologies and lack of reimbursement for posttransplant monitoring of antibodies. Nonetheless, a deleterious effect of antibody specific for mismatched donor HLA antigens has been demonstrated for nearly every type of organ and tissue that has been transplanted in sufficient numbers, including hematopoietic stem cells and, possibly, composite tissues. There is overwhelming evidence indicating that anti-HLA antibodies are involved in hyperacute, acute, and chronic rejection of organs (1). In mismatched bone marrow transplants, there is a risk of immunocompetent tissues from sensitized patients producing antibody to recipient tissues as well as a risk of recipients making antidonor antibodies. In theory, antibodies specific for any antigen on transplanted tissue should be capable of damaging the transplant. The best-known antibodies that are injurious to allografts are those specific for antigens of the HLA and ABO systems. Potential deleterious effects of other antibodies are being recognized increasingly. A discussion of those is beyond the scope of this chapter, and the interested reader is directed to two excellent reviews on this topic (2, 3). The focus of this chapter will be HLA-specific antibodies, and we will use the term donor-specific antibody (DSA) to refer to antibody(ies) specific for donor HLA antigens.
Transplantation can significantly extend the life of patients with end-stage renal disease and provide a lifesaving treatment for patients with failure of other major organs. Extending the long-term survival of these transplanted organs requires a better understanding of the mechanisms of immune injury, including humoral immunity. Alloantibodies were determined early on to be a contraindication to transplantation, but until recently the primary focus has been on HLA-specific antibodies (1). Historically, the lymphocyte crossmatch was the primary tool used to detect HLA antibodies in the sera of transplant recipients and to assess the risk of antibody-mediated rejection posttransplantation. Now, with the advent of solid-phase immunoassays, we can detect HLA antibodies at very low levels and provide a more accurate characterization of their antigen specificities (2).
Solid-organ transplantation has become an increasingly important therapeutic modality for patients with various end-stage diseases. Despite improved immunosuppression protocols, most transplant recipients face a variety of complications. Early posttransplant infection and rejection are the major causes of morbidity and mortality. Drug toxicity, chronic rejection, and malignancies are long-term complications.
Better reagents have led to an increased appreciation of the association between complement activation in transplants and poor outcome (1–4). As diagnostic markers, products of complement activation have two attractive attributes related to the fact that activation of complement proceeds through a series of enzymatic steps resulting in multiple cleavage products. First, each enzymatic step is capable of amplifying the number of molecules that are cleaved. Second, the cleavage process reveals cryptic epitopes that allow the activated products to be distinguished from the unactivated precursors. In addition, activation products from two complement components, namely, C4 and C3, have the unusual property of covalently binding to protein and carbohydrate substrates. All of these properties distinguish complement activation products from some of the molecules that activate complement, such as antibodies. Antibodies themselves are not readily detected in tissue sections because only transient binding of a small number of antibodies to tissue is required to activate exponentially larger amounts of complement.
Improved knowledge of the alloimmune repertoire, development and clinical application of new therapies, advances in surgical techniques, and effective infection prophylaxis have helped to transition transplantation from a high-risk experimental therapy to a safe clinical remedy. Nevertheless, diagnostic and therapeutic challenges remain. Here we provide an outline of the immune cascade implicated in allograft rejection, emphasize molecular protocols for characterizing gene expression strengths and patterns and for high-throughput protein and peptide analyses, and summarize molecular correlates of rejection of human kidney, heart, lung, liver, and pancreas allografts.
Both in hematopoietic stem cell transplantation (HSCT) and in solid-organ transplantation (SOT), T and B cell-mediated immunity toward nonshared HLA alleles can have a detrimental outcome. In the past decade, many studies have investigated whether natural killer (NK) cell-mediated immunity might also play a role in clinical transplantation. This work has focused on killer cell immunoglobulin-like receptors (KIRs), as these NK cell receptors interact with HLA class I molecules in an allele-specific manner. In this chapter, we describe the genetics and functions of KIRs and discuss their role in HSCT and SOT.
Chimerism testing is routinely used after allogeneic hematopoietic stem cell transplantation (HSCT) to monitor engraftment, detect relapse, identify patients with increased risk for graft-versus-host disease or graft loss, and monitor the effectiveness of therapeutic interventions. Chimerism testing can also be useful in other settings, such as detecting engraftment of donor lymphocytes after organ transplantation and determining sample identity (e.g., investigating suspected sample exchanges).
Today's clinical immunology laboratories have emerged from research laboratories as new assays became available to researchers and clinician-scientists. However, in today's hospital and reference laboratory environment, many governmental regulations and professional guidelines mandate defined education levels of individuals who oversee and perform patient testing, recommend test procedures, and define acceptable ordering, result reporting, and billing practices. Among these organizations is the Centers for Medicare and Medicaid Services (CMS), previously known as the Health Care Financing Administration (HCFA). CMS is a federal agency within the U.S. Department of Health and Human Services (DHHS) that administers the Medicare program and works in partnership with state governments to administer Medicaid. CMS also regulates billing practices and has a significant impact on the manner in which laboratories must be structured and managed. Today's successful clinical immunology laboratory practitioners must be knowledgeable about current guidelines and regulations and keep abreast of the constant changes in order to ensure compliance and professionalism in all areas of the laboratory. This chapter provides a broad overview of the major governmental agencies and regulations that impact on clinical laboratory practices. Various agencies and regulations are discussed in this chapter. To assist the reader, a list of their abbreviations is provided. In addition, an extensive list of websites is given in Table 1 to enable the reader to obtain further information regarding specific agencies, regulations, and programs.
Clinical laboratories provide vital services to physicians and patients, public health agencies, and medicolegal entities. Because laboratory test results influence patient care and medical decisions rely on accurate and timely laboratory results, clinical laboratories must ensure that the right results are obtained at the right time and for the right patient. The ability to ensure accuracy of patient results rests on developing a quality program that governs all functional aspects of the clinical laboratory.
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Doody's Review Service
Description
This eighth edition of a manual for laboratory immunology comes 10 years after the 2006 edition. It is available in a print version (5.8 pounds, 1,245 pages) and an electronic version (EPUB or PDF).
Purpose
The purpose is to provide a single, authoritative reference source for laboratory immunology. This is a most worthy objective, well met by previous editions and, true to its reputation of excellence, this edition meets it as well. The electronic versions are welcome enhancements, permitting easy portability and access and accessibility options (e.g., font size, contrast, etc.).
Audience
It is intended for those who work in the clinical immunology laboratory - from clinical laboratory scientists in training or practice, to residents or fellows in clinical immunology or laboratory medicine/clinical pathology, to attendings in practice in either discipline, and especially laboratory directors. This book would also be of interest to clinicians in the many internal medicine subspecialties treating patients with diseases with immunological pathogenesis, to better understand and therefore more appropriately use testing for diagnosis, treatment, and management.
Features
It has been 10 years since the seventh edition, which also incorporated "molecular" into the title for the first time. The revolution in the past decade in molecular-based testing methods has exploded, so this edition is much needed and welcomed. The first seven sections are devoted to general methodologies, the next nine to diseases, and the last section to laboratory management. The molecular aspects have been deftly interwoven - whether proteomic techniques, the role of the microbiome in disease, HLA typing with molecular methods, immunoglobulin gene rearrangement, or transplantation immunology, etc. There are a few omissions. Many of the disease-specific chapters contain experimental protocols, but surprisingly many lacked discussion of quality controls. Some laboratory protocols are casually referred to as "laboratory developed tests" (LDTs). LDTs have been a most controversial issue in the past few years, yet this book devotes only a single paragraph in the laboratory management chapter on the proposed regulatory requirements. While the references cited in this paragraph would provide more-than-you-could-ever-want-to-know detail about Food and Drug Administration (FDA) considerations for LDT regulation, the controversy and therefore immediate risks to accreditation and licensure are not obvious to the naive reader. An enhancement for the next edition would be cautionary alerts to readers concerning implementation of LDTs, and inclusion of quality control testing instructions for every experimental protocol.
Assessment
This is THE authoritative reference for molecular and clinical laboratory immunology. If you perform this testing or want an expert update in the field, you will want this book.
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Weighted Numerical Score: 91 - 4 Stars!
Reviewer: Valerie L. Ng, PhD MD (Alameda County Medical Center/Highland Hospital)
Review Date: October, 2016
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