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Category: Immunology
Renamed Manual of Molecular and Clinical Laboratory Immunology, the 7th edition of the Manual of Clinical Laboratory Immunology continues the tradition of serving as the guide for laboratory directors. The new edition places substantial emphasis on molecular techniques and introduces new material that reflects the significant advances and developments in the field of clinical immunology. This valuable reference will be of practical use to immunologists and will serve the needs of students, instructors, and practitioners of clinical immunology.
The book provides a concise, up-to-date review of the basic principles underlying clinical procedures, features detailed descriptions of the methodology used, and places special focus on the interpretation of laboratory findings. All existing chapters have been significantly revised and updated in the 7th edition.
The molecular theme is woven throughout the sections, with the first section of the book specifically outlining cutting-edge molecular applications soon to be introduced into the standard immunology laboratory. Timely material is introduced in chapters and sections covering transplantation, monitoring of cytokines and chemokines, multiple applications of flow cytometry, allergic diseases, and cancer.
Out of Print, hardcover, 1,340 pages, full-color insert, illustrations, index.
Major advances in proteomics and genomics have occurred and these are further impacting the field of diagnostic and clinical immunology. The completion of the Human Genome Project has fueled the field of genomics and provided the critical database from which to expand genomic studies. A vast array of proteins provides a wide range of functional elements in the immune system. Expanded evaluation of these critical products should provide new insights into the biology of the immune system and pathology of immunologic disorders. The future will depend on linking genomic and proteomic studies as partners in understanding the immune response and characterizing the underlying basis of immunologic diseases. Parallel developments in the field of genomics also impact on the field of diagnostic and clinical immunology. The information garnered by the expanding range of analytes and technologies available to evaluate the proteome and genome is fueling a new vision of scientific discovery referred to as systems biology. This integrated proteomic and genomic approach should afford far better understanding of the perturbations that lead to human disease.
There are multiple sources of variation in individual proteins and in the proteome. Posttranslational modifications of the primary translation products of mRNAs potentially lead to large numbers of structural variants of individual gene products. As proteins undergo extracellular and intracellular degradation, a complex array of peptides is generated, quite possibly even more complex than the original set of proteins. Peptide splicing has recently been identified as another mechanism that may increase peptide diversity. In order to understand many pathophysiological processes, such as prion diseases, amyloidosis, hemoglobinopathies, and apoptotic processes, simple primary sequence analysis of proteins is inadequate. Two additional challenges to protein analysis that are nearly as great as structural diversity are (i) the dynamic nature of protein concentration and (ii) the wide range of protein concentrations. Unlike the case for nucleic acids, there is no simple method for amplification of the concentration of proteins. Most methods for protein analysis have a practical analytical concentration range of only about 100to 10,000-fold, and the highest-resolution separation methods for protein analysis, such as two-dimensional gel electrophoresis, have the capability of resolving only up to a few thousand components in a single analysis.
In the context of the amazingly diverse and dynamic array of proteins in the human proteome, a relatively tiny set of proteins representing a few hundred gene products are analyzed for diagnostic purposes. Applications of mass spectrometry to analysis of plasma peptide components over the last few years suggest that there are a large number of potential diagnostic markers among these components. Further description of analysis of proteins by two-dimensional electrophoresis and peptides by mass spectrometry is present in this chapter, which describes research and potential future clinical laboratory methods. Potential types of diagnostic specimens are summarized in this chapter. Blood represents the most common specimen collected for protein analysis. There are a variety of different types of diagnostic questions that are addressed by protein analysis. The simplest is the qualitative question of whether a protein serving as a physiological marker is present or absent in a particular specimen. Binding assays represent the broadest range of functional assays for proteins in the clinical laboratory. Clinical laboratories have applied a variety of electrophoretic, chromatographic, and other separation techniques to analysis of proteins. These techniques are useful for qualitative analysis of the size, charge, associations, and other physical properties of proteins that may not be distinguished by quantitative methods such as immunoassays or functional assays. The electrophoretic method provides higher resolution than the typical methods for serum protein analysis, and it is applied in the clinical laboratory to achieve higher-resolution separations of immunoglobulins for evaluation of oligoclonal bands in cerebrospinal fluid.
In a short period, the genomics age has shifted a substantial proportion of study to the search for patterns or profiles of expression or genetic variants in specific diseases. Microarray analysis interrogates total RNA or mRNA purified from cell or tissue sources. Harvest of total RNA or mRNA is critical; technical problems or degradation fatally undermines the interpretation of expression data. Naturally, the quality control steps for ensuring that RNA is not degraded must be established, and in circumstances in which insufficient RNA is available, careful amplification of RNA can be performed during the generation of cDNA. Currently, most studies have used cDNA or oligonucleotide-based arrays to identify regions of loss of heterozygosity or gene amplifications. Chip technology was initially applied to single-base pair mutation detection, using a similar hybridization technology but with genomic DNA as a template. Currently, there are four commercial technologies available for large-scale, high-throughput genotype analysis: Affymetrix, ParAllele, Illumina, and Perlegen. The new technologies for single-nucleotide polymorphism (SNP) detection will enable studies to look at genetic variation across all genes of a pathway or biological process as well as across the entire genome. Similar to the analytical challenges of the microarray expression studies, analytical approaches will continue to evolve in SNP research. One of the major goals of the coming decade is the development of high-throughput sequence technologies.
This chapter provides a detailed discussion of the molecular techniques and equipment that are available, or under development, for use in the clinical laboratory. Molecular testing in the clinical laboratory consists of two major areas: (i) the use of DNA probes to directly detect or characterize a specific target and (ii) the use of nucleic acid amplification technologies to detect or characterize a specific target DNA or RNA. The use of DNA probe technology is discussed first in the chapter; nucleic acid amplification technology and nucleic acid sequencing are discussed later, followed by molecular arrays, a more elaborate application of probe technology. Each type of probe hybridization assay is discussed individually, as are probe amplification procedures. The primary objective of nucleic acid amplification techniques is to improve the sensitivity of assays based on nucleic acids and to eventually simplify these assays by development of automated assay formats such as real-time detection. Target amplification procedures probably provide the simplest products for post-amplification detection. In addition, dedicated procedures for the detection of amplified products following nucleic acid amplification, such as reverse dot blots, are discussed only briefly because these methods are being replaced with real-time nucleic acid amplification assays where the product is detected concurrently with the ongoing amplification. Real-time nucleic acid detection systems are also discussed in this chapter. Only through education and the ability to meet new challenges will clinical immunologists be able to control the use of molecular techniques and practice of molecular diagnostics in the clinical immunology laboratory.
This is the introductory chapter to the section Immunoglobulin Methods. The section examines the topics of immunoglobulin production by gene rearrangements, the measurement of immunoglobulins, identification of monoclonal protein products by serum protein electrophoresis and immunofixation, detection of oligoclonal bands in cerebrospinal fluid, and characterization of cryoglobulin, cryofibrinogen, and pyroglobulins. The chapter states how the other chapters(7th to 11th chapters) in the book are organized. The chapter by Kipps (chapter 7) examines the genes that code for immunoglobulins. Warren (chapter 8) overviews how the immunoglobulin structure is related to its function and the utility of serum viscosity evaluations. In chapter 9, Keren and Humphrey review the technical details and clinical applications of serum and urine protein electrophoresis. Katzmann and Kyle (chapter 10) present a thorough review of characterization of monoclonal gammopathies in serum and urine by immunofixation and immunosubtraction. Gorevic and Galanakis (chapter 11) present the state of the art for detecting and measuring cryoglobulins, cryofibrinogenemia, and pyroglobulins.
Resolution of immunoglobulin structure has revealed how Immunoglobulin molecules can have such great diversity in antigen-binding activities while maintaining conserved effector functions, such as complement activation. IgG is the predominant antibody produced during a secondary immune response. IgG molecules can penetrate extravascular spaces and cross the placental barrier to provide immunity to the fetus. IgA antibodies are the primary antibodies in saliva, tears, and colostrum and in the fluids of the gastrointestinal, respiratory, and urinary tracts. IgM is the predominant class found during a primary immune response. IgD molecules are thought to function as B-cell membrane receptors for antigens and may help in the recruitment of B cells for specific antigen-driven responses. Plasma IgE levels may increase (5 to 20 times the baseline) in parasitic infections and children with atopic diseases. Duplication of an immunoglobulin VH gene(s) results in some haplotypes’ having identical immunoglobulin VH genes belonging to distinct loci, each possibly differing from their respective alleles by one or more nucleotide base substitutions. Immunoglobulin class-switching recombination (CSR) occurs in or near the α switch region upstream of the μ gene and any one of the switch regions of the other heavy-chain isotype genes. Resolution of the junctional sequences in the rearranged immunoglobulin genes expressed by a tumor can provide a specific tumor marker. This marker can be used to examine for any tumor-derived immunoglobulin gene fragments amplified by PCR performed on genomic DNA of lymphoid tissue.
Quantification of intact immunoglobulins has proven useful in the evaluation of patients with suspected immunodeficiency syndromes, B-cell and plasma cell neoplastic diseases, allergic conditions, and chronic inflammatory and autoimmune disorders. This chapter provides a review of immunoglobulin structure, important because of its direct relevance to quantitative immunoglobulin measurement. It presents an overview of assay methods, issues related to quality control and assurance, and test validation, as well as a brief discussion of the clinical application of immunoglobulin measurements. Finally, the chapter addresses both technical and clinical aspects of viscosity measurement.
This chapter reviews basic principles of electrophoresis, the types of apparatus that are available, quality control and quality assurance procedures, and costs involved with the procedure and provides a wide variety of patterns with recommended interpretations. Electrophoresis of serum and urine in the clinical laboratory takes advantage of the fact that each protein has its own unique structure. The first studies of serum protein electrophoresis were performed entirely in a fluid-based system devised by Arne Tiselius, winner of the 1948 Nobel Prize in Chemistry, called moving boundary electrophoresis. An early-morning void collected into a container without preservative is adequate for screening for the presence of monoclonal free light chains. Each protein electrophoresis gel should have an internal control sample run on it. The percentage of protein in each fraction should be recorded and compared day to day. Good quality assurance requires coordination of all available laboratory information for the patient. A file is set up for the patient the first time that an M protein is detected in the serum or in the urine. In patients with cirrhosis, synthesis of hepatocyte-derived proteins such as albumin and transferrin is decreased. In protein-losing enteropathy, damage to the gastrointestinal tract such as occurs in gluten-losing enteropathy (celiac disease), the serum demonstrates findings similar to those of the nephritic syndrome with hypoalbuminemia, hypogammaglobulinemia, and occasionally elevated levels of α2 macroglobulin. The urine immunofixation is compared to the urine protein electrophoresis to determine which band is the monoclonal free light chains (MFLC).
This chapter focuses on qualitative methods for the assessment and characterization of clonality. The methods include nondenaturing agarose gel electrophoresis (AGE) with immunofixation, capillary zone electrophoresis (CZE) with immunosubtraction, and isoelectric focusing with immunoblotting. 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 immunosubtraction electrophoresis (ISE) are diagnostic tools used for the identification of monoclonal gammopathies and, conversely, for the confirmation of polyclonal hypergammaglobulinemia. Isoelectric focusing with immunoblotting is a cerebrospinal fluid (CSF) diagnostic test for the identification of oligoclonal bands (OCB) in multiple sclerosis (MS). The monoclonal gammopathies include a spectrum of diseases, from ones that may have little clinical significance to ones that may be rapidly fatal. These include multiple myeloma (MM), Waldenström’s macroglobulinemia, smoldering MM, monoclonal gammopathy of undetermined significance (MGUS), primary systemic amyloidosis (AL), lymphoproliferative diseases, and plasmacytomas. High-resolution agarose gel protein electrophoresis (PEL) and CZE fulfill the requirements for a screening procedure for detection of monoclonal proteins. Monoclonal free light chains are uncommon in MGUS and are associated predominantly with MM or AL. The assessment of urine samples by IFE is similar to serum IFE. The major difference is the need to concentrate urine samples to achieve an appropriate protein concentration. The concentration of immunoglobulins is increased in the CSF of patients with inflammatory diseases of the central nervous system, such as MS, neurosyphilis, and acute inflammatory polyradiculoneuropathy.
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). Mixed cryoglobulins are cold-precipitable rheumatoid factors (RFs), with the serum often being positive when standard assays for IgM antiglobulin activity are used. Although many clinical laboratories offer cryoglobulin determinations, rarely is testing rigorously carried out, and there is considerable interlaboratory variability. A search for cryofibrinogenemia may be dictated by unexplained thrombohemorrhagic coagulopathy or cold-dependent purpura; it may also be dictated by the finding of characteristic pathology, occurring in various affected organs, as for example an occlusive thrombotic diathesis due to eosinophilic deposits within vessel lumina, extending into the intima, which may be associated with a granulomatous vasculitic component. Cryofibrinogenemia can be screened by cryoprecipitating or freeze-thawing plasma collected in citrate that contains an inhibitor of thrombin generation and is most convincing in the absence of coexisting cryoglobulinemia. Recognition of the laboratory phenomenon of pyroglobulinemia has importance as a potentially confounding factor for heat-based assays used to inactivate complement or to measure fibrinogen levels. Proper identification of a pyroglobulin as being a monoclonal component may in turn lead to the diagnosis of macroglobulinemia or plasma cell dyscrasia.
Complement provides a fast-acting mechanism for the identification and removal of foreign substances before the more specific arms of the adaptive immune system can come into play. Many clinical laboratories have shied away from the analysis of complement, due in large part to the liability of the system and the special handling that samples require if the results are to be reliable. The symptoms of inflammation associated with complement activation are due to a limited number of biologically active complement split products that are produced by the enzymatic cascade. Neutrophils, monocytes, macrophages, eosinophils, and mast cells can be enticed to perform many of their tricks (e.g., chemotaxis and mediator and enzyme release) by the complement fragments. The major control of the alternative pathway (AP) occurs through the action of two proteins that stop the cleavage of C3.
Familial hemolytic uremic syndrome (HUS) was first described in 1956; both autosomal dominant and recessive forms of inheritance have been reported. The chapter discusses three pieces of evidence which suggested that complement genes apart from the gene for factor H were involved in the pathogenesis of atypical HUS. Structurally, membrane cofactor protein (MCP) consists of four alternatively spliced isoforms that coexist on most cells. The extracellular domain is composed of four control protein modules (CCPs). Western blot, flow cytometry, cell surface labeling, and pulse-chase analysis all showed that the mutant protein was retained intracellularly. Noris et al. described a family in which two siblings were affected by HUS. Mutation screening in both revealed a heterozygous 2-bp deletion in MCP exon 7, which encodes CCP4. The functional effect of this mutation is similar to the deletion mutation and results in half of the normal level of cell surface expression of MCP. The two techniques most frequently used for mutation scanning are denaturing high-performance liquid chromatography (DHPLC) and single-strand conformation polymorphism (SSCP). DHPLC is based on heteroduplex analysis in which mutant and wild-type sequences present in a PCR product are heated and allowed to reanneal slowly. This results in the formation of two heteroduplexes and two homoduplexes. MLPA (multiplex ligationdependent probe amplification) is a relatively new method for detecting deletions and duplications in a simple two-stage procedure and gives dosage information for up to 45 exons in one test.
The complement system has a crucial role in innate immune defense against invading microorganisms and can be activated by the classical pathway (CP), the alternative pathway (AP), and the mannose-binding lectin (MBL) pathway (MP). The CP is activated by binding of C1q to, e.g., immunoglobulins present on microorganisms or by direct binding to apoptotic cells. The AP can be directly activated by invading microorganisms. The MP is also directly activated, via carbohydrate moieties present on the surface of invading microbes. For assessment of the functional activity of the classical and alternative pathways, hemolysis of erythrocytes by complement activation via either the CP (CH50) or the AP (AP50) is used in most laboratories. The methods to assess pathway activity of the CP, AP, and MP are enzyme-linked immunosorbent assay (ELISA)-based. All three assays are delivered as one ELISA system. Blood samples are to be collected under sterile conditions in red-top tubes, without serum separator. A positive control is provided in the kit. Frozen sera is partially thawed by briefly placing them in a 37№ C water bath with gentle mixing. With the combined assay, the functional activity of the three pathways of complement activation can be assessed. With the use of combined ELISA system, the three pathways for complement activation can be assessed at the same time.
Events in the mannan-binding lectin (MBL) pathway of complement activation involve the binding of MBL to patterns of carbohydrate structures presented on the surface of microorganisms and the following activation of the proenzymes of the complement system. The protocols for MBL antigen assay are described in this chapter and are useful for routine quantification of MBL and evaluation of the MBL pathway activity in clinical samples. The assays are based on the robust, highly sensitive and reproducible time-resolved immunofluorometric assay (TRIFMA). TRIFMAs are similar to enzyme-linked immunosorbent assays (ELISAs), with the only difference being the type of labeling of detecting molecules. Different approaches have been used to measure the MBL concentration in plasma or serum. The first is based on a modification of the conventional sandwich ELISA, in which microtiter wells are coated with anti-MBL antibody and then incubated with dilutions of plasma or serum and the amount of bound MBL is measured by using europium-labeled anti-MBL antibody (MBL antigen assay). The second assay quantifies MBL on the basis of its lectin-binding activity (lectin assay), in which microtiter wells are coated with mannan instead of anti-MBL antibody. The MBL antigen assay is more sensitive (down to 2 ng of MBL/ml of plasma) than the lectin assay (10 ng/ml).
This chapter examines the advantages and disadvantages of various reagents to complement as applied to the diagnosis of different types of rejection in transplants. Activation of the complement cascade produces many split products that do not bind to tissues or that are released from tissues. These fluid-phase products can be detected in plasma, serum, urine, and bronchoalveolar lavage samples. Enzyme-linked immunosorbent assay kits are available to quantitate soluble split products in fluids. Hyperacute rejection is the most unambiguous example of complement-mediated injury to transplants. In hyperacute rejection, complement activation is initiated by large amounts of antibody binding to antigens on the endothelial cells of the transplanted organ. The most characteristic features of hyperacute rejection can be predicted from our knowledge of complement. Complement can be a useful adjunct in diagnosing and directing treatment of transplant rejection. Currently, the tissue-associated final split products of C4b and C3b, namely, C4d and C3d, are the most useful markers of antibody-mediated rejection. Both C4d and C3d offer the advantages of being produced in large amounts and binding covalently to tissues. Monoclonal antibodies to neoantigens on C4d and C3d that are not accessible in the unactivated precursors are available. Although these markers have provided significant diagnostic advances for organ transplantation, appropriate interpretation requires additional information, including testing for circulating antibodies to donor antigens and correlation with clinical evidence of graft dysfunction.
This is the introductory chapter to the section Flow Cytometry. In an effort to capture the burgeoning field of clinical flow cytometry, the chapters presented in this section are divided into two parts. The first part focuses on current application, which encompasses updates in established procedures as well as new procedures that have been recently adopted. The second part is more innovative, with applications combining molecular methodologies and flow cytometry. This section provides a quick review of clinical flow cytometry applications. Stem cell transplant is no longer a rare procedure performed only at highly specialized institutions. With the advent of cord blood banking, new safer stem cell transplant protocols, and numerous new clinical trials, accurate enumeration of stem cells in a variety of products has become an essential procedure for an increasing number of institutions. The X-linked hyper immunoglobulin M (IgM) syndrome is a combined immune deficiency resulting from mutations in the genes encoding the CD40 ligand (CD154). A diagnostic flow cytometry screening test has been developed for the rapid detection of CD40 ligand expression abnormalities. Currently, the most commonly utilized laboratory methods for laboratory diagnosis of allergy involve the detection of allergen-specific IgE. Improved standardization of the allergens used for in vitro testing combined with improvements in the procedures will lead to an increased adoption of this testing modality in the near future.
This chapter provides a broad description of the principles of flow cytometric immunophenotyping, provides specific methods for lymphocyte enumeration with particular emphasis on CD4 T-cell counting, and reviews quality assurance issues that improve the accuracy and reproducibility of overall immunophenotyping results. Samples for immunophenotyping are prepared by incubation with fluorochrome-labeled monoclonal antibodies (MAbs), and red blood cells are removed by lysis to prepare the sample for analysis on the flow cytometer. MAbs are created by the fusion of B-cell tumors and primary B cells previously selected to make antibodies to only one epitope of a specific antigen. The lyse/no-wash (LNW) method permits single-platform (SPT) absolute counting. A second protocol is provided for laboratories that perform more advanced immunophenotyping, using MAbs that must be used in lyse/wash (LW) sample procedures prior to running on the cytometer. The use of a sample handling device which can be loaded with sample tubes inside the biosafety cabinet and attached to the flow cytometer for automated processing reduces sample handling and samples can be safely vortexed throughout sample acquisition. CD4 T-cell levels are important measures for staging human immunodeficiency virus (HIV)-1 disease and predicting disease progression. In HIV-1-infected adults the CD4 T-cell absolute count is the most useful measure of disease progression, while in children CD4 T-cell percentage is preferred because of high variability in lymphocyte counts.
This chapter focuses on four less common clinical applications of flow cytometry: paroxysmal nocturnal hemoglobinuria (PNH), T-cell receptor Vβ (TCR Vβ) analysis, fetal hemoglobin (Hgb F) detection, and platelet surface marker and functional testing. Three PNH red blood cell (RBC) phenotypes are recognized due to variations in genetic defects that can result in PNH. These are types I, II, and III, which exhibit normal, moderate, and severe complement sensitivities, respectively. Classical clinical tests for PNH are aimed at demonstrating the presence of RBCs that are exceptionally sensitive to the hemolytic action of complement compared to normal RBCs. Flow cytometry is becoming the preferred method for assessing blood samples for the presence of PNH clones. In recent years, monoclonal antibody reagents specific to various TCR-β family V regions have become available. This method offers greater sensitivity and specificity than classical tests in identifying patients with PNH. Structural and functional defects in platelets can lead to a variety of bleeding disorders. Development of commercially available antibodies to relevant platelet antigens permits the discrimination between activated and resting platelets and permits platelet receptor quantitation. Clinical diagnosis is supported by monitoring uptake of mepacrine (a fluorescent molecule that is rapidly taken up and localized to dense granules) into dense granules of platelets and its loss with dense granules release upon stimulation. Idiopathic thrombocytopenic purpura is primarily a disease of increased peripheral platelet destruction, with most patients carrying antibodies to specific platelet membrane glycoproteins, resulting in splenic sequestration and phagocytosis by mononuclear macrophages.
The medical indications for the use of flow cytometric leukemia and lymphoma immunophenotyping in a clinical setting continue to be defined, but three general areas have been identified. The general principles and procedures for instrument setup and specimen handling are similar to those used for lymphocyte immunophenotyping; however, in this chapter, emphasis is laid on areas of particular importance to leukemia and lymphoma immunophenotyping. Optimization and standardization of instrument performance on a daily basis are very important in the immunophenotypic analysis of leukemia and lymphoma. Immunophenotyping of leukemia and lymphoma may be performed on any specimen from which a single cell suspension can be generated, including peripheral blood, bone marrow, lymph node, body fluids, and tissue. Peripheral blood and bone marrow specimens must be anticoagulated; EDTA, heparin, and acid-citrate-dextrose (ACD) are the anticoagulants most frequently used. The detection of hematopoietic malignancies by flow cytometry relies on the principle that neoplastic cells express antigens in patterns recognizably different from those of normal hematopoietic cells. In B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), the abnormal B cells express the T-cell-lineage-associated antigen CD5 in combination with dim CD20, dim CD22, dim surface light chain, and moderate to bright CD23, with little to no expression of FMC7. The determination of clonality by flow cytometry has recently been described for T-cell or NK-cell neoplasms, utilizing T-cell receptor Vβ subset or killer inhibition receptor subset antibodies, respectively.
Recent studies have indicated that umbilical cord blood (CB) represents a rich source of CD34+ hematopoietic stem/progenitor cells, and consequently, there has been a proliferation of CB banks worldwide. Currently, the enumeration of CD34+ stem/progenitor cells by flow cytometry represents the most clinically useful surrogate marker of graft adequacy and provides crucial information to the transplant physician. While flow cytometric enumeration of CD34+ cells represents the most clinically useful assay of graft assessment, the assays that were initially developed were insufficiently robust, and interinstitutional variability in particular was problematic. An increasing array of ex vivo manipulations has been developed to engineer the graft to suit specific clinical requirements. Included in the latter are positive selection techniques to purify CD34+ cells and negative purging techniques to remove residual tumor cells in the autologous setting, or T lymphocytes in the allogeneic setting. Finally, there is widespread interest in the development of clinically useful ex vivo expansion methodologies and gene therapy protocols. At present, the authors feel that the methodologies based on the original International Society of Hematotherapy and Graft Engineering (ISHAGE) protocol and updated in Current Protocols in Cytometry (CPC) represent the most accurate and flexible protocols currently available to both clinical and research laboratories for the analysis of the increasingly wide variety of normal and abnormal hematopoietic samples.
The first classification of the acute leukemias in 1976 relied exclusively on the evaluation of cell size, granularity, nuclear shape, cytoplasmic appearance, cytochemical reactions, and dysplastic features of cells surrounding the “leukemic blast.” In selected cases, researchers have even succeeded in not only elucidating but also reversing the oncogenic mechanism, such as in acute promyelocytic leukemia (APL). APL has become the paradigm for the ultimate goal of leukemia diagnosis: to be able to tell an oncologist what targeted therapy a patient is a candidate for, based on the detection of a specific genotype. Some antibodies invariably stain all cells from a given lineage but vary markedly in their intensity of staining between normal and malignant cells, suggesting variable antigen densities (e.g., those of CD20 and CD22 on normal versus chronic lymphocytic leukemia [CLL] B lymphocytes); for other antigens, the fraction of cells binding the antibody will contain the diagnostic information (e.g., the percentage of CD34+ or CD117+ cells in any acute leukemia). Acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), or chronic myelogenous leukemia (CML) tissues should be cultured for 24 h without mitogens before processing. Samples from CLL require stimulation by B-cell mitogens (B-cell CLL) or T-cell mitogens (T-cell CLL) for cell division to occur. The principle of targeted therapy, as attractive and promising as it is, requires much more understanding of transforming molecular events and perturbed signaling pathways than the simple administration of indiscriminately cytotoxic chemotherapy.
T cells play essential effector and regulatory roles in adaptive immune responses. For accurate measurements of T-cell immunity as it exists in vivo, the act of in vitro expansion creates at least two significant problems. First, the efficiency of the expansion is highly variable and therefore the results are semiquantitative at best. Second, the stimuli required for in vitro expansion of specific T cells unavoidably alter their phenotype, inducing changes in the patterns of cell surface molecules involved in crucial functions such as cell adhesion and trafficking, as well as potentially altering effector functions such as cytokine secretion profiles. The major histocompatibility complex (MHC) tetramer version was the first to be described and has the largest publication record behind it. Identification of antigen-specific T cells by antigen-binding methods requires no assumptions with respect to the potential functions of the target cells. By far the most popular fluorophore labels for MHC tetramers are the algae-derived phycobiliproteins R-phycoerythrin and allophycocyanin. MHC tetramer stains are nearly always combined with an anti-CD3 antibody as well as either a CD8 antibody (for class I tetramers) or a CD4 antibody (for class II tetramers). Human leukocyte antigens (HLA) tetramer staining for flow cytometry can be performed using relevant single-cell suspensions from any source. A flow cytometer with a minimum of three fluorescent channels is required for collection of data from HLA tetramerstained cells. In addition to the flow cytometer, a variety of standard laboratory equipment-tabletop centrifuges, pipettes, vortex mixers, microscopes and hemacytometers is required.
The ideal situation in diagnostics would be if one could in a sensitive and specific manner detect certain disease-causing genes, including infectious agents, without destroying the cells. From a diagnostic perspective, the detection of genes and proteins within a cell would need to be high throughput on a user-friendly platform capable of an expanded menu. This chapter describes multiple approaches of performing molecular biology in a cell. It also describes how to use the assays to gain a better understanding of disease processes and ultimately how the assays can be adopted in clinical laboratories to provide the most comprehensive information on response to a variety of therapies. The powerful combination of single-copy detection in cells combined with the high-throughput cellular analysis platform of flow cytometry led to the elucidation that human immunodeficiency virus type 1 (HIV-1) infected enough cells to account for the severe immune destruction leading to AIDS. In situ self-sustained sequence replication (IS-3SR) is based on the use of primers with attached RNA polymerase initiation sites and the combination of three different enzymes in the same reaction mixture (DNA polymerase, RNase H, and RNA polymerase), resulting in accumulation of target mRNA through the combination of reverse transcription, DNA synthesis, and in vitro transcription. The development of HIV-1 entry inhibitors, most notably the CCR5 inhibitors, will present an exciting opportunity for cell-based diagnostics.
This is the introductory chapter to the section Functional Cellur Assays. A number of advances in basic immunology, molecular biology, and cellular physiology have resulted in major developments in cellular immunologic assays relevant to clinical and diagnostic immunology. The application of these techniques in research and clinical laboratories has led to their high sensitivity and specificity. These techniques afford the clinical immunology laboratorians, the clinical allergists and immunologists, and physicians in allied specialties with assays and techniques which make possible the precise diagnoses of primary and secondary disorders of the immune system. The chapters presented in this section incorporate newer diagnostic assays that have come into use. Approaches for molecular diagnosis of chronic granulomatous disease have been presented. There is direct application of these assays in clinical medicine related to primary and secondary immune deficiency, autoimmunity, hematology, rheumatology, transplantation, and a wide array of clinical disorders.
This chapter provides a brief discussion of the molecular mechanism of action and a survey of the different implementation tools, as well as test interpretation. In addition, the clinical implications of delayed-type hypersensitivity (DTH) skin testing in disease diagnosis and screening (i.e., tuberculosis) and the monitoring of specific disease progression (i.e., human immunodeficiency virus [HIV]) will be reviewed. DTH skin testing with two antigens (yeast cell suspension and polysaccharide antigens) from the same strains of Candida can produce discordant results in up to 20% of individuals. In the assessment of the cell-mediated immunity (CMI) response to recombinant gpl60 immunization in asymptomatic HIV-infected patients, the sensitivity of DTH skin testing compared to LPA was 75%. In addition, DTH skin testing can also aid in the diagnosis of many bacterial and fungal infections such as tuberculosis, leishmaniasis, histoplasmosis, blastomycosis, and aspergillosis. DTH skin testing provides a practical tool in the assessment of CMI. It can be used to establish defects in CMI, predict progression of and monitor HIV disease, test responses to vaccines, and diagnose bacterial and fungal infections. For valid interpretation of DTH testing, the skin test placement and accuracy of the skin test reading as well as various health factors need to be taken into consideration. Lastly, DTH correlates well with the more specific in vitro lymphoproliferative assay (LPA), and it remains the recommended initial screening tool for CMI on the basis of its ease of use and inexpensiveness.
The use of cryopreserved peripheral blood mononuclear cells (PBMC) for the studies of immune reconstitution in human immunodeficiency virus (HIV)-infected patients permits the selection of samples from well-characterized study subjects. The goal of the cryopreservation is to freeze and thaw the PBMC without compromising cell viability. Both glycerol and dimethyl sulfoxide (DMSO) can provide this function, but only DMSO has been extensively studied for cryopreservation of PBMC. Other critical steps of the cryopreservation procedure include inhibition of cell metabolism while in the presence of DMSO, rate-controlled freezing to avoid cell dehydration, rapid thawing, and resuspension in serum-containing medium, which protects the cells against osmotic trauma. Lymphoproliferative responses measure mainly CD4-dependent immune responses. Severely impaired CD4 responses confer broad immunodeficiency. Natural killer (NK) cell activity, in contrast to cytotoxic T lymphocytes (CTL), is more liable to freezing and thawing. Enumeration of PBMC subsets by flow cytometry provides important information on the immune capacity of the host. As such, flow cytometry-based immune phenotyping is a standard procedure in the evaluation of immunodeficiency disorders. The distribution of major surface markers, such as CD3, CD4, CD8, CD19, and CD14, is essentially unchanged by cryopreservation. Definition of the T-cell receptor repertoire has become increasingly prevalent in studies of the maturation and senescence of the immune system. The two techniques seem to yield similar results when performed with fresh and cryopreserved PBMC.
The assay most widely utilized is the enzyme-linked immunospot assay (ELISPOT). The ELISPOT was first described in 1983 as an alternative to plaque-forming assays for the detection of antibody-secreting cells. This assay may be adapted for use with synthetic peptides, including overlapping peptide pools. In an effort to increase the sensitivity of ELISPOT, several groups have developed modifications of the standard protocol involving the addition of exogenous cytokines, costimulatory antibodies, or antigen-presenting cells. The ELISPOT is relatively new, and considerable methodological variation exists among laboratories. As a positive control for cytokine release, many laboratories rely on polyclonal stimuli. However, these reagents may give rise to a nearly confluent lawn of spots. Statistical methods for evaluating ELISPOT results and determining the appropriate positive cutoff have varied considerably between laboratories. A summary of statistical methods reported in the recent literature is given. Standardization and validation of any bioassay is a lengthy process and will require many multicenter studies. As ELISPOT readers continue to improve and statistical methods achieve broader acceptance, the trend towards increasing standardization is likely to accelerate rapidly within the next few years.
During the process of differentiation in the thymus, T cells undergo rearrangement of their T-cell receptor (TCR) genes and mature into single positive cells of either the CD4+ or the CD8+ phenotype. This chapter updates methods for assessment of the TCR Vβ repertoire in peripheral blood by CDR3 length spectratyping by multiplex PCR and with monoclonal antibodies (MAbs) by flow cytometry. Whole blood is collected by venipuncture into a tube containing an anticoagulant such as heparin, acid-citrate-dextrose, or EDTA. The reverse transcriptase (RT) enzyme uses RNA dependent DNA polymerase activity to synthesize cDNA from an RNA template. The cDNA may be synthesized with oligo(dT) or TCR-specific C-region (Cβ-14) primers. Bands in the sequencing gel are converted into peaks by the ImageQuant software and give information similar to that of the fluorescent PCR but without the CDR3 sizes that are computed by the automated DNA sequencer. A reduction in the number of peaks compared to that of the control range represents restrictions in the repertoire. Southern blotting can be used for assessment of TCR Vβ and Jβ segments. The extracted DNA is treated with a set of restriction enzymes. The products of digestion are separated by agarose gel electrophoresis, blotted onto a nylon membrane, and probed with a set of labeled Jβ probes.
This chapter focuses on defining the chronic granulomatous disease (CGD) subtype and identifying the specific genetic defect. Before genetic analysis of CGD patients is performed, it is advisable to identify the affected protein component to pinpoint the defective gene. The molecular defects that cause the X91, A22, and A67 forms of CGD are highly heterogeneous in nature, with many of them being family specific. In addition to confirming the CGD subtype, the identification of specific mutations can provide important information for detecting or confirming carriers among patients’ family members and for prenatal diagnosis in affected families. Once genomic DNA has been isolated, single-strand conformational polymorphism (SSCP) analysis can be used initially to examine the 13 exons of the gene, in order to identify the region likely to contain a mutation. More than 90% of all A47 CGD patients analyzed to date are homozygous for the deletion of GT from the normal GTGT at the beginning of exon 2 (ΔGT/ΔGT genotype).
With the implicit understanding that monocytes and granulocytes represent distinct cell lineages, this chapter reviews laboratory procedures which can be utilized to assess their functions and phenotypes. In the chapter, assays which have proven to be adaptable for routine clinical use or have potential for increased utility in a clinical context are reviewed. Eosinophils and basophils complete the peripheral myeloid cell subsets. Basophil function is briefly described in the context of allergy in the chapter. Physical isolation procedures may also lead to the selective loss of specific monocyte subsets. Phagocytosis is a complex physiological process involving the engulfment and internalization of material bearing appropriate surface molecules. The measurement of phagocytosis is confounded by the difficulty of differentiating bound from internalized material, regardless of whether this process is evaluated by microscopy or flow cytometry. The chapter talks about a procedure that assesses phagocytosis by flow cytometry and differentiates between surface-bound and internalized particles. It describes a method that is commercially available as a kit (Orpegen) and includes an optional procedure to exclude from analysis cells with bound bacteria that have not been internalized (i.e., phagocytosed).
Programmed cell death is a normal, physiological event which occurs without inflammation, so nontargeted bystander cells are not harmed. It is the body’s mechanism for eliminating cells which are no longer needed or potentially injurious. The study of apoptosis is being increasingly applied to many different cell types (e.g., hepatocytes, neurons, cardiac myocytes, etc.). The methods discussed in this chapter refer to the analysis of apoptotic cells in the sample of interest by utilizing flow cytometry, which has emerged as a technology well suited to quantifying dying cells. The subdiploid assay is highly recommended as an initial assay for a simple evaluation of apoptosis. The major advantages of this method include its ease, similarity to familiar assays of the cell cycle, and low cost, and in addition, following flow cytometric analysis, samples can be spun down onto slides and examined by fluorescence microscopy. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method is based on the enzyme-mediated insertion of nucleotides into DNA strand breaks which are present in apoptotic cells. Apoptotic cells containing active caspase-3 or -7 will exhibit an increased fluorescence intensity compared to that of the live cells in the same sample. The potential to treat human disease via manipulation of apoptotic pathways holds great promise, and as the elucidation of both protective and inductive cell death signaling mechanisms continues, such possibilities approach realization.
In chickens, blood lymphocytes may be identified as recent thymic emigrants (RTEs) by the use of antibodies that react with the cell surface marker chT1. PCR quantification of T-cell receptor (TCR) rearrangement excision circles (TRECs) derived from TCR β chain locus rearrangement has been described and has been used to provide additional evidence that the differentiation of T cells with a diverse TCR repertoire continues until late adulthood. The quantitation of TRECs may be performed with unsorted cells or with subpopulations of cells purified by fluorescent cell sorting, magnetic bead separation, panning, or other techniques. In view of its high yield and relative simplicity, magnetic cell sorting with CD4 and CD8 microbeads and positive selection columns is often used. High TREC concentrations have been associated with changes in T-cell populations after the transplantation of thymic tissue in patients with DiGeorge syndrome and, in some cases, after highly active antiretroviral therapy (HAART) for human immunodeficiency virus (HIV) infection. Moreover, recent data suggest that TREC values may prove to be a useful predictor of disease progression for HIV infection.
In most cases, natural killer (NK) cells activity is determined in the peripheral blood and only rarely in the target tissue, largely because it is difficult to isolate NK cells from diseased tissues in humans. Many of the newer cytotoxicity assays are based on flow cytometry and are nonradioactive. In combination with flow cytometry for the enumeration of cells expressing the NK-cell phenotype, the assay can provide a quantitative estimate of NK-cell function during human health and disease. The NK activities of fresh and cryopreserved peripheral blood mononuclear cells (PBMC) from the same individuals have been compared and have encountered differences as large as 100%, although PBMC from certain individuals can be cryopreserved without such losses in cytolytic function. NK cells can be subdivided into two functionally distinct populations on the basis of CD56 and CD16 expression on the cell surface. Biologic variability controls: fresh PBMC from the same healthy donors are obtained repeatedly at various time intervals and used to chart the biologic variability in NK-cell activity over time. Measurements of NK activity are difficult to perform in a clinical laboratory setting. They require considerable expertise, constant vigilance, and extensive quality control and quality assurance.
A greater understanding of the immune response, particularly of cell-mediated immune responses in malaria, tuberculosis (TB), human immunodeficiency virus (HIV), and cancer patients, will lead to improved vaccine design. Currently, in vitro analyses of cellular immune responses are being used for assessments of the efficacy of therapeutic and prophylactic vaccines, for clinical diagnosis, and for studies of immune regulation. This chapter was compiled by authors who have been involved in HIV vaccine testing for 15 years and have been collaborating with or have worked in laboratories in both developed and developing countries. When conducting cellular immunology assays, the integrity of the peripheral blood mononuclear cells (PBMC), especially the cellular membranes, is critical for success. In a separate experiment, the authors assessed the effect of the time from collection to processing on the proportions of lymphocyte subsets (total T cells, CD4 and CD8 T cells, B cells, and NK cells). The standard LPA measures antigen-induced cell division. Cells (usually PBMC) are incubated in the presence of various concentrations of an antigen (specific) or mitogen (nonspecific) stimulus of interest. The percentages of responders to mitogens, recall antigens, and HIV antigens were consistent for comparisons of fresh and cryopreserved PBMC. CD8+ T lymphocytes are able to mediate a variety of effector mechanisms and may provide the basis for protective immunity against a diverse array of infectious pathogens and tumors. The influences of host genes, health and nutrition status, and disease burden of patients are taken into consideration by conducting studies in-country.
This chapter reviews signaling events associated with T-cell activation. Crystal structure analysis has confirmed that the molecular specificity of the T-cell receptor (TCR) is conferred by the solvent-exposed membrane-distal variable regions of the α and β chains. The earliest biochemical events elicited by T-cell activation are the phosphorylation of proteins in the TCR-CD3 complex and the activation and interaction of Syk and Src family protein tyrosine kinases (PTKs). Importantly, LAT is palmitoylated, which targets it (and the molecules it recruits) to glycolipid-enriched microdomains known as lipid rafts. Recruitment of the tyrosine-phosphorylated TCR to lipid rafts is a critical step in T-cell activation and presumably concentrates the downstream signaling machinery in close proximity, facilitating enzymatic activity and molecular scaffold formation. The reorientation and redistribution of the cytoskeleton constitute an important consequence of T-cell activation, since they are closely linked to functional outcome. Defects affecting the termination of T-cell activation and growth may result in systemic autoimmune disease. The sensitivity of the quantitative fluorescence is based on the use of the standard curve as described in the standardization section. The combined use of flow cytometry and evolving technologies, such as gene microarray systems and proteomics, will tremendously increase the understanding in the field of T-lymphocyte activation and signaling.
The function of the humoral immune response differs depending on the class of antibody produced and the differentiation state of the B cell. Primary defects in the humoral components of the immune response are usually recognized by clues as frequent development of infections early in life and difficulty in clearance of infections. The most direct measure of in vivo B-cell function is immunoglobulin secretion by plasma cells. Serum immunoglobulins are most commonly measured by automated nephelometry. Antibodies provide protection to the host in various ways. Antibodies can neutralize toxins (e.g., tetanus toxin), neutralize viruses, prevent the adhesion of bacteria to the host cells, kill bacteria in the presence of complement, and opsonize bacteria for phagocytes. Generally, the complement-mediated bactericidal mechanism does not kill gram-positive bacteria, although there are exceptional cases. This mechanism is primarily relevant in the study of antibodies to gram-negative bacteria, which have thin walls. Opsonophagocytosis is the primary protective mechanism of antibodies against gram-positive bacteria. Various methods have been developed to measure the opsonizing capacity of antibodies in vitro. The classical approach is to perform an opsonophagocytic killing assay. In this assay, the bacteria are opsonized (coated) with antibodies and complement. The increased use of functional assays has shown the importance of standardization.
This chapter is an introduction to Cytokines and Chemokines. In the clinical laboratory, cytokine assessment has been used to monitor disease progression and activity. In addition, the increasing use of cytokines and cytokine antagonists as therapeutic modalities requires the measurement of cytokine levels to determine the pharmacokinetics of the administered molecule. In the research laboratory, the measurement of cytokine gene expression is currently being explored with the hope that this approach will offer clues to better define the mechanisms of cytokine action in disease processes. A section encompasses reviews on cytokines, chemokines or adhesion molecules.
This chapter explores both traditional enzyme-linked immunosorbent assay (ELISA) and the newer multiplexed assays. For each of the methods, the author examines the technology, instrumentation, and data analysis. The sequential ELISA still has several pitfalls. First, the sample volume necessary is still greater than that used in most multiplex assays. Second, the amount of time required to measure each one of the cytokines is not reduced in the sequential ELISA, so there is no cost savings with regards to time. Third, if there is potential cross-reactivity of the antibodies, then measurement of the first cytokine will result in an overestimate of the amount of that cytokine present and measurement of the next cytokine in the sequence will result in an underestimate of the amount of that cytokine present. A recent paper described a novel method for rapidly detecting cytokines by capillary electrophoresis. Bead array assays are rapidly becoming the rage in cytokine measurement. There are several commercial vendors marketing the bead array assays The major limitation is obtaining antibody with sufficient specificity in order to prevent cross-reactivity. Finally, the volume of sample required to measure multiple cytokines is extremely small. Multiplex cytokine assays for measuring cytokines are rapidly becoming standard across the world. A clear indicator of the enthusiasm for these assays may be found in the large number of companies which are presently producing and manufacturing multiplex cytokine kits.
This chapter describes the optimized methods for cytokine flow cytometry (CFC) that offer increased throughput and robustness. The basic principle of the CFC assay is that whole blood or peripheral blood mononuclear cells (PBMCs) are activated with a specific antigen in the presence of a secretion inhibitor such as brefeldin A (BFA) or monensin for a short duration. The samples in the plates can be acquired using a multiwell-plate loader connected to the flow cytometer. Dynamic gating strategies that account for sample-to-sample staining variability can simplify data analysis and improve the reproducibility of the results. The procedural details discussed in the chapter include recommendations for sample type and handling, choice of antigen(s) and plates, and the gating strategy for this improved CFC format using cytomegalovirus (CMV) pp65 peptide mix as a model antigen. Gating of flow cytometry data for analysis is a frequent source of assay variation, especially in rare-event assays such as CFC where antigen-specific responses may be as low as 0.1%. In situations where large numbers of samples need to be evaluated, e.g., in vaccine immune response-monitoring studies, it is desirable to minimize time and errors that may occur in assay setup and processing. The authors have optimized the process of using lyophilized antigens and lyophilized antibody cocktails for CFC assays of both PBMC and whole blood.
During the last decades, analysis of cytokines and cytokine receptor research have gathered much interest. Numerous methods have been developed for the detection and quantification of cytokines and cytokine receptors, which may be detected at either the protein or the mRNA level. This chapter focuses on improvements of the recently developed real-time quantitative PCR that has been used in our laboratory for a couple of years in comparison to other available techniques. The methods described allow quantification of minute amounts of mRNA in small samples due to the exponential amplification of the target sequence and the generation of fluorescent molecules that are detected during the amplification reaction. The performance of amplification reactions is affected to a large extent by the concentrations of magnesium ions and oligonucleotide primers. Usually, specificity of primer annealing increases with decreasing magnesium ion concentration. With real-time PCR, specificity in terms of amplification of only the correct sequence is not the major goal, since hybridization and cleavage of the internal probe guarantee specificity of target quantification. A major advantage of the real-time PCR is its application as a high-throughput analytical system in routine diagnostics and research, as, in contrast to other conventional PCR-based techniques, no post-PCR processing of the reaction products is required.
This chapter describes the methodological approaches to studying the role of chemokines and chemokine receptors in the physiology of immune and inflammatory responses. Chemokines share the common function of attracting leukocytes to sites of an inflammatory or immune response. The G protein-coupled cell surface receptors (GPCRs) as signal transducers of chemotaxis appear to be highly conserved in evolution, being present on amoebae and slime molds, and are involved in the signaling of chemotaxis in these simple eukaryotes. Activation of chemokine receptors is usually accompanied by a transient rise in the level of intracellular calcium. The attraction of leukocytes to sites of inflammation and infection is an essential component of the host response to disease. Chemokines and chemokine receptors have been shown to be an integral part of this process and have been implicated in the pathophysiology of many infectious diseases and inflammatory disorders. The recruited monocytes and T lymphocytes are thought to play a key role in the pathogenesis of atherosclerotic plaque, and thus the molecular signals that attract these cells into the lesions are likely important for lesion formation. The majority of cells recruited into the synovium are neutrophils and mononuclear leukocytes that presumably help propagate the inflammation and joint destruction. Chemokine expression in the lung or blood could be used as a reliable noninvasive marker of inflammation in the airways and thus would be of great value in the management of asthma.
The goal of laboratory monitoring for cytokine therapies is to maximize the efficacy and minimize adverse effects of these agents. The optimal monitoring strategy will depend on the characteristics of the specifics of the therapy, but general strategies can be identified. In IFN-β therapy for multiple sclerosis (MS), many of these general strategies have been utilized with significant benefit to MS patients. In other cytokine based therapies (CBTs), monitoring has not been as extensively utilized. There are two major types of laboratory measures used to monitor CBTs: biomarkers of disease and cytokine-induced molecules. Binding-antibody (BAb) assays are generally faster, more sensitive, and more inexpensive than other assays. Enzyme-linked immunosorbent assay (ELISA) technology can be used, which allows for the screening of hundreds of samples in a few hours in an automated fashion. CBTs are a subclass of biologicals that have profound biological effects, many of which are poorly understood. These drugs are recent additions to therapy, and consequently, we are still relatively early in a growth curve about their optimal use. Monitoring of their effects is going to be important for many reasons. One of the major reasons is that these therapies tend to be highly immunogenic and the optimal approach for identification of antidrug antibodies and their potential to neutralize drug effects needs to be identified. Another reason is the high cost of these biologicals, which makes optimal use critical.
This is the introductory chapter to the section Immunohistology and Immunopathology. The contemporary practice of anatomic pathology is intertwined with and unthinkable without the contributions of immunohistochemistry. The instrumentation not only to perform immunohistochemical assays but also to evaluate histopathological slides in general is becoming more automated, which has shortened turnaround times. Enhancements in the technology will increase the utilization and value of the assays discussed in the chapter. The increased understanding and application of molecular methods will undoubtedly alter the practice not only of pathology but also of many other subspecialty areas, including surgery, gynecology, oncology, hematology, and gastroenterology.
This chapter presents current, practical methods that are applicable for performance of quality immunostains in the modern automated immunohistochemistry (IHC) laboratory. The cross-links are believed to be responsible for masking antibody-binding epitopes and to be a major cause of lack of sensitivity in paraffin section IHC. In spite of its inherent detrimental effects on antigenicity and IHC, a proper and consistent protocol for fixation with neutrally buffered formalin is imperative for high-quality IHC. Paraffin tissue sections for IHC are usually cut at a thickness of 4 to 6 μm. In order to retain sections on the glass microscope slide throughout the staining process, it is necessary to use slides that have been specially treated to increase their adhesive properties. Most antibodies used in a clinical IHC laboratory are commercially available and have well-characterized specificities. For analyte-specific reagents, it is assumed that good manufacturing practices have been followed and that the antibody will likely perform as advertised. Antigen retrieval enables antibodies that might previously have performed only in frozen tissues to be reactive in fixed tissues and has greatly expanded the number of antibodies that can now be applied to paraffin-embedded tissue.
This chapter provides an overview of several examples of molecular biology applications in this rapidly advancing field. Not all present biomarkers can be detected in tissues that are routinely available in pathology laboratories. There is an ongoing effort to adapt techniques that work well with fresh, unfixed tissues to specimens such as formalin-fixed paraffin-embedded needle biopsy material and cytology smear material. Ligase chain reaction and similar techniques are presently used for the detection of microorganisms in tissues by labeling the oligonucleotides with organism-specific sequences. This process can also be used for detection and quantitation of the expression of specific genes in tissues. The study of familial adenomatous polyposis has demonstrated the presence of adenomatous polyposis coli gene mutations in 5q21. The study of molecular pathways of carcinogenesis has highlighted various molecules that are now therapeutic targets. A relatively simple method of examining hundreds of tissue sections in one experiment is the use of tissue microarrays (TMAs). PCR-based methodologies with the advantage of using fresh, frozen, or formalin-fixed tissues or cells are faster and less labor-intensive and have become the first-line methods in clinical laboratories for clonality testing. The majority of lymphomas and leukemias can be further classified by their characteristic chromosomal lesions. Similar to those associated with hematopoietic malignancies, most genetic abnormalities associated with soft-tissue tumors are chromosomal translocations resulting in novel fusion proteins.
This chapter highlights the basic principles of immunostaining for light microscopy in brief and discusses the diagnostic and prognostic applications of immunostaining. Improved standardization of fixation methods, automated immunostaining, and image analysis are likely to improve the global interpretation of tissue subjected to immunohistochemistry. The areas in which immunostaining might be used for such applications may be categorized as follows: differential diagnosis, evaluation using therapeutic predictive markers, evaluation using prognostic markers, evaluation for dysplasia and malignancy, and intraoperative evaluation. Her-2/neu protein is expressed in the cell membrane and can be demonstrated by immunohistochemistry as a marker with a membranous immunostaining pattern. In cases with equivocal immunohistochemistry results, gene amplification can be demonstrated by the use of labeled nuclear probes with in situ hybridization, either fluorescent in situ hybridization or chromogenic in situ hybridization.
In many skin diseases, demonstration of immunoglobulin (Ig) or complement deposition in specific structures in the dermis and epidermis is an essential criterion for accurate diagnosis. For the skin, the focus is primarily on the immunofluorescence findings in bullous and connective tissue disorders, vasculitides, and other mucocutaneous conditions, for which this immunohistochemistry technique can provide useful diagnostic information. This chapter emphasizes the common immunofluorescence techniques used for diagnosis and interpretation with kidney and skin biopsy specimens. For skin specimens used for immunofluorescence studies, the proper choice of a biopsy site is critical to maximize the probability of obtaining diagnostic information. In most if not all cases of bullous skin diseases, namely, those autoimmune in nature, biopsy specimens should be obtained from perilesional tissue. For the diagnosis of pemphigoid, pemphigus, linear IgA bullous disease, epidermolysis bullosa acquisita (EBA), and dermatitis herpetiformis, the tissue sample should be taken from inflamed but unblistered skin. Most diagnostic studies using direct immunofluorescence techniques are performed with unfixed frozen tissue sections, since many antigens can be altered or destroyed by fixation. Indirect immunofluorescence is used to detect circulating autoantibodies with tissue specificity. These include anti-glomerular basement (anti-GBM) and anti-tubular basement antibodies in renal disease and bullous pemphigoid and pemphigus antibodies in skin disease. Photography is the only permanent record one has of the immunofluorescence findings. Many immunopathology laboratories are now using a digital camera interfaced with a computer to capture fluorescence images for documentation of results.
This is the introductory chapter to the section Infectious Diseases caused by Bacteria, Mycoplasmas, Chlamydiae, and Rickettsiae. The chapter describes a variety of methods and their applications for detecting infections caused by bacteria, mycoplasmas, chlamydiae, rickettsiae, and bartonellae. In addition to antibody detection methods, procedures for assessing cellular immune responses to selected pathogens (e.g., Mycobacterium tuberculosis) are discussed in the chapter. Both in vivo assays (e.g., skin testing) and in vitro assays (e.g., gamma interferon secretion) for detecting cell-mediated immune reactivity are presented. Molecular methods for pathogen detection are increasingly performed under the auspices of the clinical immunology laboratory.
The M protein is a major virulence factor of group A streptococci. Antigenic differences within the hypervariable region at the N terminus constitute the basis for the Lancefield serological classification for group A streptococci. Identification of M types is accomplished using a precipitation reaction between M-protein antigen and type-specific antiserum. The antisera against the M-protein antigens are produced with whole-cell streptococcal (killed) vaccines used to immunize rabbits. M-protein antigen is extracted from streptococci using Lancefield's hot hydrochloric acid method. Rapid streptococcal antigen tests have overcome the inherent overnight delay associated with the culture method for identification of group A streptococci and have allowed clinicians to make management decisions based on laboratory data shortly after examining the patient. Nephelometry has been introduced as a simple, rapid procedure for quantitative measurement of anti-streptolysin O (ASO) titers. The principle of nephelometry is based on the ability to measure the rate of increase in light intensity scattered from particles suspended in solution as a result of complexes formed during an antigen-antibody reaction. The streptococcal antihyaluronidase determination is an enzyme neutralization test in which the antigen used is streptococcal hyaluronidase elaborated by the bacteria. It should be emphasized that the most common clinically used streptococcal antibody tests (ASO and anti-DNase B) provide evidence only for a preceding group A streptococcal infection.
Estimation of antibody concentrations and measurements of functional antibody are critical to one's understanding of polysaccharide (PS)-induced immunity. Information on quantitative immunoassays for anti-PS antibodies is presented in this chapter. In an assessment of naturally occurring levels of antibody to encapsulated bacteria and evaluation of the immune response to PS or conjugate vaccines, one should consider both antibody quantitation and measures of biological (functional) activity. Quantitation of anti-PS antibody is important, because as the type-specific antibody concentration increases, the risk of invasive disease on a population basis due to that bacterial strain decreases. The enzyme-linked immunosorbent assay (ELISA) is now the most commonly used method for quantitation of antibody concentration. The presence or induction of bactericidal antibody is predictive of protection against meningococcal disease. The major problem that Neisseria meningitides presents is its ability to cause outbreaks and epidemics. The pneumococcal PSs are poorly immunogenic and fail to induce protective antibody levels in children <2 years of age. For this reason conjugate vaccines were developed against the 7 to 11 most common pediatric pneumococcal types. In Nepal, where the attack rate in individuals 5 to 44 years of age was 16.2/1,000, the Vi PS vaccine given as a single 25-μg dose had an efficacy of 75% measured 17 months after vaccination. This study clearly showed that serum antibodies to the Vi PS were sufficient to protect against typhoid fever.
Corynebacterium diphtheriae and Clostridium tetani are responsible for important diseases. Tetanus is caused by introduction of C. tetani from soil through contaminated wounds, while diphtheria results from airborne infection from infected cases. In both instances it is a potent exotoxin that is responsible for clinical disease. The toxin of C. tetani is so potent that the lethal dose may not be of sufficient strength to stimulate antibody production. In both instances formalin-inactivated toxins have been used for universal immunization. For children they are combined with pertussis vaccine, and adults are given both together at a reduced dose of diphtheria toxoid. The exotoxin produced by C. diphtheriae is the most important pathogenic factor. Although reliable methods of measuring antitoxic neutralizing antibodies have existed for many years for tetanus antitoxin, regular clinical laboratories rarely maintain the capability of determining tetanus antitoxin levels because they are of no use in diagnosis or treatment of the acute disease. The mouse neutralization assay is the most widely used, and this method is described in this chapter in detail. Injection of tetanus toxoid, which is one of the most effective immunizing agents in use today, regularly evokes antitoxic antibodies. There are few indications for the determination of tetanus antitoxin levels. The procedure for titration of tetanus antitoxin is based on the capacity of tetanus antitoxin to protect mice from death after subcutaneous injection of tetanus toxin.
The families Enterobacteriaceae and Vibrionaceae encompass a heterogeneous group of gram-negative organisms. The organisms chosen for discussion in this chapter are recognized as major public health concerns because of their significant contribution to morbidity and mortality in both children and adults in less developed and industrialized countries. Cytotoxicity is usually examined under the microscope or can be quantified by colorimetric methods. Positive samples should be retested in the presence of Shiga toxins (Stx)-specific antibodies to confirm the specificity of the cytotoxic effect. The optimal concentration of antigen should be determined by standard enzyme-linked immunosorbent assay (ELISA) checkerboard titration, coating plates with different amounts of antigen and testing them with known positive and negative control sera during the standardization of the assay. Diagnosis of acute enterotoxigenic Escherichia coli (ETEC) infections requires the detection of labile toxin (LT), ST, or toxin genes in isolated strains. Typical enteropathogenic E. coli (EPEC) strains carry both a chromosomally encoded (the locus of enterocyte effacement (LEE), which mediates a complex signal transduction phenotype on the target epithelial cell, and the EPEC adherence factor plasmid (EAF), which encodes the principal EPEC adhesin.
The CagA protein, which is highly immunogenic, is one of the most widely studied virulence factors of Helicobacter pylori. The cagA gene is one of the genes in the cag pathogenicity island. A variety of techniques are available for the diagnosis of H. pylori infection. In this chapter the tests are discussed generically rather than listed specifically. In an attempt to eliminate the need for venipuncture, diagnostic tests have been developed for the detection of IgG and IgA antibodies in saliva and urine. Studies using urine-based enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies against H. pylori have been conducted. The most important factor in using immunological methods for identifying VacA-specific antibodies by ELISA is the antigen preparation used to detect circulating antibodies. The availability of recombinant VacA protein will help to make the VacA ELISA more widely available. In summary, ELISAs for detection of H. pylori-specific antibodies are sensitive, specific, and cost-effective in untreated individuals. The major limitation of such tests is the lack of a rapid, clear serologic response to eradication of bacterial infection. Molecular tests have been developed which preclude culture and allow direct, sensitive detection of H. pylori and specific virulence factors thereof in a variety of biological specimens. Much work remains to be done to standardize molecular assays so that they can be applied generally to the diagnosis of H. pylori infection and eradication.
Determination of the levels of antibody to Legionella pneumophila was one of the first methods used to diagnose Legionnaires’ disease and was especially valuable when culture diagnosis was at a primitive stage. This chapter describes antibody testing because it is still very widely used and is of major importance for epidemiologic and research studies. Patients infected with some Legionella serogroups may not demonstrate antibody to other serogroups. About 40% of people who eventually seroconvert have agglutinating antibodies detected during the first week of infection. Detection of specific IgA antibodies may also increase the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA). The sensitivity of the kits for measurement of antibody to L. pneumophila SG2 to SG6 is unknown, but they do appear to be reasonably sensitive in comparison to the indirect immunofluorescent-antibody (IFA) method (70 to 90%) for the estimation of SG1 antibodies. Cross-reactive antibody is occasionally found in patients with non-Legionella infections. Up to 25% of people with Campylobacter jejuni enteritis have cross-reactive antibodies to L. pneumophila. However, as many as 10% of patients with tuberculosis had cross-reactive antibodies to other Legionella serogroups and species. It is unclear whether this absorption removes only nonspecific antibodies and what criteria should be used to evaluate the serologic results of testing before and after the use of this blocking antigen. Pontiac fever is an acute, nonfatal febrile illness associated with Legionella but not proven to be caused by it.
Syphilis is the most common spirochetal disease in the United States, with the most recent estimate being 32,871 newly reported cases in 2002. For the spirochetal diseases, direct dark-field microscopic examination often provides an immediate means of presumptive diagnosis. The direct fluorescent-antibody test for Treponema pallidum (DFA-TP) examination of tissue and body fluids is particularly valuable in the diagnosis of congenital syphilis. Subtyping for T. pallidum done at the Centers for Disease Control and Prevention is based on two genes that exhibit intrastrain variability, the genes encoding the acidic repeat protein (arp) and the T. pallidum repeat (tpr) protein. A diagnosis of leptospirosis should also be considered in cases of unexplained jaundice, aseptic meningitis, and fever of unknown origin. Definitive diagnosis of leptospirosis is made by isolation of leptospires from tissue or body fluids, but leptospires grow slowly on initial isolation and culture is therefore often not useful in patient management. The diagnostic tests discussed in this chapter are the microscopic agglutination test (MAT), which is the standard against which all other serologic tests for leptospirosis are evaluated, and two screening tests, the immunoglobulin M (IgM) dot enzyme-linked immunosorbent assay (ELISA) and the IgM ELISA. The MAT detects agglutinating antibodies and is considered the standard reference test for the diagnosis of leptospirosis.
Lyme disease is the most common tick-borne bacterial disease in the northern hemisphere. The disease is caused by three genomic groups or genospecies of Borrelia burgdorferi sensu lato: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. Only B. burgdorferi sensu stricto is known to cause Lyme disease in North America, whereas all three genospecies are responsible for the disease in Europe. Serology is the most useful type of laboratory test that is widely available to support a clinical diagnosis of Lyme disease. Positive serologic test results, however, should not be used by themselves to establish this diagnosis. Laboratory testing of samples from such patients also is not recommended, since testing will result in more false-positive results than true positives. Serum specimens first should be evaluated by a sensitive enzyme immunoassay (EIA) or immunofluorescent assay (IFA). During the first month of infection, both immunoglobulin M (IgM) and immunoglobulin G (IgG) antibody responses should be determined. A strain used as an antigen in a serologic test should express appropriate amounts of the immunoreactive proteins of diagnostic interest. Positive-control serum samples from patients with well-characterized Lyme disease are required, preferably four samples that possess a range of anti-B. burgdorferi antibody levels from low to high.
An inverse relationship between cell-mediated and humoral immunity has been described for different forms of tuberculosis and is the essential discriminator between lepromatous and tuberculoid leprosy. Studies of mice in which the genes for gamma interferon or IL-12 and their receptors have been deleted have conformed the importance of these cytokines in resisting the development of tuberculosis after aerosol challenge. Historically, antigens specific for Mycobacterium tuberculosis have been identified by using pre-absorbed hyper-immune serum or monoclonal antibodies. A meta-analysis of studies, which included random allocation of subjects for BCG vaccination and measured the number of cases and/or deaths due to tuberculosis, also demonstrated significant benefit from BCG vaccination in preventing active tuberculosis. The overall relative risks for tuberculosis and for death due to tuberculosis were 0.49 and 0.29, respectively, in those who received BCG vaccination compared to non-vaccinated subjects. Four controlled trials have shown that BCG vaccination is effective against the development of leprosy, and in one trial in which both tuberculosis and leprosy were studied, benefit was confined to the prevention of leprosy. The WHO recommends BCG vaccination for children in areas where HIV infection and tuberculosis are common unless the children have symptoms of AIDS.
This chapter discusses the advantages and disadvantages of current molecular and serologic diagnostic techniques for mycoplasmal infection and ureaplasmal infection. Established approaches to the detection of Mycoplasma pneumoniae, Ureaplasma, Mycoplasma genitalium, and Mycoplasma hominis infections are discussed. A majority of the discussion in the chapter focuses on M. pneumoniae, but many of the same principles and limitations of detection also apply to the other members of the Mollicutes. In addition, the extent to which newer commercial serologic assays for M. pneumoniae will cross-react with M. genitalium or other mycoplasma was not established with certainty, but this seems less likely to be a problem than with complement fixation (CF). An in-depth discussion of various types of commercial M. pneumoniae antibody assays and a comparative analysis of them based on published studies is available in a recent review. Perhaps more than for any other mycoplasma, molecular biology-based techniques have played the critical and indeed defining role in diagnosis of M. genitalium infections. The molecular biology-based techniques and considerations are essentially the same as for the other mycoplasmal species. The most common target gene is 16S rRNA, and both multiplex PCR and real-time PCR assays have been developed. Because of the relative ease of cultivation, standardization and validation of the PCR assays can be more easily achieved.
Chlamydiae are obligate intracellular bacteria, which cause many diseases in animals and humans. This chapter focuses on the diagnosis of human infections. Chlamydia trachomatis genital infections are associated with many syndromes, including cervicitis, urethritis, pelvic inflammatory disease, infertility, and ectopic pregnancy in females, and urethritis, proctitis, and epididymitis in males. Serovars can be distinguished by serology or by genetic typing of the major outer membrane protein (MOMP) gene. Antigen detection using an enzyme immunoassay (EIA) was used before the advent of molecular tests and is still the most common nonculture detection test for C. trachomatis. Nucleic acid amplification tests (NAATs) can be used to define the infected patient gold standard to evaluate chlamydia diagnostic tests. Symptomatic patients should receive a pelvic examination, with a sample taken for a NAAT, as recommended by the Centers for Disease Control and Prevention (CDC) as the test of choice. The species C. pneumoniae was established in 1989 based on antibody reactivity, EB ultrastructure, and DNA homology. C. pneumoniae causes 10% of pneumonia and 5% of bronchitis and sinusitis cases in adults. Avian strains (Chlamydia psittaci) are virulent to humans and cause severe pneumonia accompanied by systemic symptoms known as psittacosis. Transmission occurs by the airborne route, either by direct contact with birds or by the inhalation of dust contaminated with excreta of infected birds.
The order Rickettsiales consists of the family Rickettsiaceae and the family Anaplasmataceae. The family Rickettsiaceae contains the genus Rickettsia and the genus Orientia. Members of the family Rickettsiaceae are short rods or coccobacilli, and members of the family Anaplasmataceae are small pleomorphic cocci. The family Bartonellaceae consists of two genera, one of which, Bartonella, includes human pathogens belonging to the α2-group proteobacteria. Currently, the principal techniques used for the serodiagnosis of rickettsial diseases are probe-based immunoassays. Immunoprobe-based tests include the indirect fluorescence assay (IFA) and its micromodification, the micro-IF (MIF) test. The MIF test is still considered as the gold standard. Other immunoprobe tests include immunoperoxidase assays (IPAs); enzyme-linked immunosorbent assays (ELISAs), in which the antigens are adsorbed either onto the well of a microtiter plate or onto nitrocellulose in a dot blot or slot blot configuration; and immunoblot assays (IBAs). IFAs and IPAs require the whole bacterium as the antigen. The remaining immunoprobe assays use either highly purified rickettsiae or an extract. The development of real-time PCR-based diagnosis and rapid PCR-based diagnostic methods has been the focus of much attention in many research laboratories. Recently, a protein-based method for the detection of the small-cell variant protein A (ScvA) plus other protein markers comprising a Coxiella fingerprint via matrix-assisted laser desorption-time of flight (MALDI-TOF) mass spectrometry has been developed.
In the last decade, there has been a dramatic resurgence of pertussis, particularly in adolescents and adults, likely as the result of a waning of vaccine induced immunity. Detection of the etiologic agent is the optimal method of making a laboratory diagnosis; however, with Bordetella pertussis infections, the nature of the infection and the natural history of the infection make this difficult. Because of the difficulties with the test, DFA staining should only be used in conjunction with culture; some national surveillance systems do not accept DFA results as laboratory confirmation of pertussis infection. PCR is also less affected by antimicrobial effects than is cell culture, permitting laboratory diagnosis even after effective antibiotics have been initiated. The measurement of pertussis agglutinins is one of the oldest assays used for determining antibody titers against B. pertussis. Widely used in the early and mid-20th century, particularly in the Medical Research Council clinical trials that demonstrated the efficacy of whole-cell pertussis vaccines, pertussis agglutinins correlated well with population immunity to pertussis but did not predict individual protection. Pertussis toxin (PT) is the most important pertussis antigen and is responsible for many of the biological activities of B. pertussis. Proponents of the Chinese hamster ovary (CHO) cell neutralizing assay argue that the assay measures biologically active antibodies and thus might correlate better with protective antibodies. Improvements in serological diagnosis have the greatest potential for improving the laboratory diagnosis of pertussis, particularly as the epidemiology shifts from young infants and children to adolescents and adults.
This chapter focuses on methods that are currently in use within Laboratory Response Network (LRN) laboratories and on commercially available tests that have been approved by the U.S. Food and Drug Administration (FDA) and thus are widely available. The chapter includes four sections, covering culture, molecular, antigen, and antibody detection methods for the diagnosis of anthrax. Supportive laboratory tests including the LRN PCR assay, immunohistochemical (IHC) staining of tissues, and anti-protective antigen (anti-PA) immunoglobulin G (IgG) detection by an enzyme-linked immunosorbent assay (ELISA) are discussed in this chapter. LRN real-time PCR assay was also used during the 2001 bioterrorism-associated anthrax outbreak to detect Bacillus anthracis in environmental samples and clinical specimens to confirm anthrax diagnosis when the isolation of B. anthracis failed due to the initiation of antimicrobial drug treatment. Antibodies have been used successfully to detect the vegetative and spore forms of B. anthracis. A section covers the following four methods: a direct fluorescent-antibody assay (DFA), time-resolved fluorescence (TRF) assays, an immunochromatographic assay, and IHC assays. In addition, quantitative ELISAs and the toxin neutralization assay (TNA) have also been used in vaccine-animal challenge studies to show that levels of anti-PA antibodies are a significant predictor of survival. Recently, a commercially available test was FDA approved to qualitatively measure anti-PA antibodies in human serum (QuickELISA Anthrax-PA kit). Current research interests and funding will continue to yield novel technologies and new methods for the detection of B. anthracis and the diagnosis of anthrax in the future.
This is the introductory chapter to the section Mycotic and Parasitic Diseases. Parasitic diseases are historically defined as infectious illnesses caused by unicellular protozoa or multicellular helminths distinct from viral, bacterial, or fungal etiologic agents. Parasites encompass a heterogeneous group of organisms with extremely diverse biologies. Protozoa are usually a few micrometers in size, whereas worms are typically centimeters to meters in length. Tissue-dwelling protozoa are often intracellular parasites. Both protozoan and helminth pathogens have complex life cycles, often with two or more developmental stages present in the host during infection. Immune responses directed against a single stage may be circumvented by parasite differentiation. The diagnosis of mycotic infections cannot always be definitively addressed by culture or histology.
The diagnosis of parasitic infections is definitively made by the identification of parasites in host tissue or excreta. The detection of antibodies can be very useful as an indicator that an individual has been infected with a specific parasite. A positive result for a person with no exposure to the parasite prior to recent travel in an area where disease is endemic may be interpreted as indicating recent infection. In general, the detection of antibodies to parasitic diseases indicates only infection at some indeterminate time and not necessarily an acute or current infection. The detection of specific immunoglobulin M (IgM) and IgA antibodies may be of value in determining the approximate time of initial infection with Toxoplasma gondii, but it is not recommended for any other parasitic disease. The diagnosis of human intestinal protozoa depends on microscopic detection of the various parasite stages in feces, duodenal fluid, or small intestine biopsy specimens. Antibody and antigen detection tests for parasitic diseases are discussed. For all antibody detection tests for parasitic diseases, sera or plasmas are acceptable specimens. Fresh or preserved stool samples are acceptable for antigen detection testing with most kits. Antibody detection kits that are available commercially in the United States for parasitic diseases other than toxoplasmosis are tabulated and discussed in this chapter.
This chapter reviews the most extensively evaluated or routinely used tests for the serodiagnosis of mycotic infections. Pulmonary aspergilloma occurs when Aspergillus fumigatus or other Aspergillus species colonize preexisting cavities of tuberculosis, sarcoidosis, or bronchiectasis. Systemic candidiasis should be suspected if serial serum specimens show an increase in titer or an increase in the number of reactive bands detected over time. Serologic tests may also be employed to determine the potential clinical significance of Candida species recovered from various body sites. Enolase, a 48-kDa cytoplasmic antigen of Candida albicans, is a potentially useful diagnostic marker of invasive candidiasis. Tests to detect anti-Coccidioides immitis antibodies are of proven usefulness for the diagnosis and management of coccidioidomycosis. Antibody detection is of value for the diagnosis of cryptococcosis during the early stages of the disease, before antibodies are neutralized by the large amount of capsular antigen released during evolution of infection. The Histoplasma polysaccharide antigen EIA (HPA test) is a microtitration plate-based double-antibody sandwich enzyme immunoassay to detect antigenuria and antigenemia in disseminated histoplasmosis. A major diagnostic precipitin is consistently found in the sera of patients with paracoccidioidomycosis, which reacts with a soluble, specific Paracoccidioides brasiliensis antigen. Invasive fungal infections studied included aspergillosis, candidiasis, fusariosis, and trichosporonosis.
This is the introductory chapter to the section Viral Diseases. The reliance on molecular techniques has expanded significantly; they have come into common usage for numerous virus infections. In addition, the number of viral agents recognized to have an etiologic role in human infections continues to expand. As we continue to develop new vaccines, antiviral drugs, more sophisticated technologies to detect viruses more rapidly, and better reagents, we have increased our reliance on the diagnostic laboratory. The major pathogenic virus chapters have all been updated and there is considerable useful information on the latest developments in direct detection methods for viruses. Rapid viral diagnosis, using a combination of direct detection, serology, cell culture, and molecular diagnostics, is now a reality for most virus diseases. This section provides an overview of the standards for viral diagnostics as well as the latest developments in the discipline.
This chapter presents an overview of rapid viral diagnosis, focusing on the advantages and limitations of common methods used, the factors involved in test selection, the validation and monitoring of test performance, and the critical importance of sample collection. The methods used for rapid viral diagnosis can be generally grouped according to the following strategies: (i) direct detection of viral proteins, nucleic acid, or particles in clinical specimens; (ii) biologic amplification of infectious virus in cell culture followed by detection of viral antigens in cultured cells; and (iii) detection of an immunoglobulin M (IgM) antibody response to viral infection. Advantages and limitations of these methods are listed in this chapter. With the exception of electron microscopy (EM), rapid tests detect only the specific agent(s) sought. In general, the most rapid approach is to detect virus directly in clinical samples, rather than wait either for virus to grow in culture or for an antibody response to occur. Lastly, it has been shown that when physicians call the laboratory for advice prior to sample collection, the isolation rate doubles. The choices for rapid viral diagnosis continue to increase. The number and types of techniques chosen will vary with the individual viruses, laboratory expertise, and clinical needs and will continue to change as both test methods and antiviral therapy evolve and improve.
Several viruses such as herpes simplex virus (HSV), respiratory syncytial virus (RSV), influenza virus, and cytomegalovirus (CMV) were able to infect cells cultured in vitro, but the time for recognition of the cytopathic effects (CPE) produced by these agents ranged from a few days to several weeks. In addition to the age of cells, condition of cell monolayers, and specificity of monoclonal antibodies for immediate-early or early viral antigens, other important variables include incubation temperature, number of shell vial cell cultures used per specimen, centrifugation, type of specimen (urine, blood, bronchoalveolar lavage (BAL) fluid, tissue), quality of fluorescence equipment, chemical pretreatment of cell mono-layers, and technical experience with the assay to subjectively evaluate specific results. Several comparisons have demonstrated that the shell vial assay is as sensitive as (for HSV and respiratory tract viruses [adenoviruses, parainfluenza virus types 1, 2, and 3, enterovirus, influenza A and B virus, measles virus, mumps virus, and RSV]) or even more sensitive than (for CMV) the recovery of these viruses in conventional tube cell cultures, which may require several days to weeks for recognition by CPE. The diagnostic laboratory may always find a use for cell cultures; however, the next level of test performance in the clinical laboratory will be formatted for the automated extraction and quantitation detection of target nucleic acids. Amplified nucleic acids will be monitored in real time by thermocycling instruments designed to be used in routine biosafety level 2 laboratories.
Human infections with herpes simplex virus (HSV) type 1 (HSV-1) and HSV-2 are ubiquitous throughout the world. The most frequent manifestation of primary HSV-1 infection is gingovostomatitis in young children, with pharyngitis occurring more commonly in adolescents. The seroprevalence of HSV-2 has increased more than 30% in recent decades, and the majority of seropositive persons are unaware that they are infected. Humans produce antibody to the structural components of the virus, the envelope, capsid, and internal proteins, as well as soluble nonvirion antigens specified by the virus. The only human herpesvirus that shows significant cross-reactivity with HSV-1 and HSV-2 is varicella-zoster virus (VZV). Paired serum samples from patients with recent VZV infection who have preexisting HSV antibody may sometimes show a rise in the level of antibodies to HSV-1 and HSV-2 in nonspecific tests. It is advisable to use several different HSV-susceptible cell lines, such as HEp-2, Vero-E6, and RD, to maximize the chance for successful culture. Enzyme immunoassay (EIA), indirect hemagglutination assay, immunoblotting, immunofluorescence assay (IFA), and the neutralization (NT) test have had the widest application for serodiagnosis and serosurveys. Radioimmunoassays are infrequently used because current EIA and immunoblotting procedures fulfill the same purpose and avoid the problem of using and disposing of radioisotopes. In studies directly contrasting the performance of PCR with culture, direct fluorescent-antibody assay, and other techniques, PCR is invariably more sensitive and at least comparably specific.
Laboratory diagnosis of varicella-zoster virus (VZV) infection requires the identification of the virus or one of its products in skin lesions, tissues, or fluids from the patient. Techniques include isolation of the virus in tissue culture, direct immunofluorescent staining of cells obtained from lesions, and detection of the virus genome by techniques based on PCR. Vesicular fluids, crusts that have been ground in water, or skin lesion sections can be examined by electron microscopy (EM). The authors had experience of successful identification and typing of VZV strains from environmental swabs. Recent studies indicate that while multiple polymorphisms were identified between vaccine and wild-type strains, detection of the sequence polymorphism at genome position 106262 is the most reliable method for discriminating the vaccine strain from wild-type VZV. Perhaps the most important concern in the use of PCR is the quality of the template to be amplified. Serologic testing is most often requested for determining susceptibility to VZV in outbreak settings, but on occasion it is needed for diagnosis. Assay formats used for simple determination of VZV serostatus include enzyme-linked immunosorbent assay (ELISA), membrane fluorescence, and latex bead agglutination. Each of these test formats has limitations, which are discussed in this chapter. The authors recently documented cases of varicella in health care workers who were incorrectly identified as VZV seropositive by the latex bead agglutination method. Despite VZV immunoglobulin (VZIG) prophylaxis, approximately two-thirds of infants exposed to maternal varicella around the time of delivery will become infected.
Epstein-Barr virus (EBV) is generally acquired by oral transmission of virus in saliva. EBV has been linked with several human tumors including Hodgkin’s disease, non-Hodgkin’s lymphoma, nasopharyngeal carcinoma, some T-cell lymphomas, and leiomyosarcomas and B-cell lymphomas, especially in the central nervous system, in immunocompromised persons and organ transplant recipients. The Paul-Bunnell heterophile antibody associated with acute infectious mononucleosis agglutinates sheep and horse erythrocytes, among others, and is adsorbed by beef erythrocytes but not guinea pig kidney cells. The determination of IgM antibody to viral capsid antigen (VCA) (IgM-VCA) is the most valuable serologic procedure to diagnose acute EBV infection; antibody panels that do not include IgM-VCA are not as dependable for detection of acute EBV infection. High levels of IgA-VCA and IgA-EA are also found in persons with nasopharyngeal carcinoma, including the early asymptomatic stages. In these patients, tumor activity and response to cancer therapy may be monitored by serial IgA-VCA and IgA-EA determinations. Monoclonal antibodies directed against components of EBNA-1, EBNA-2, LMP-1, EA, VCA, and BZLF1 are commercially available, although the commercial EBNA-1 monoclonal antibody has not proved satisfactory for direct antigen detection. In situ hybridization is the most specific of these four molecular biological methods because it permits direct evaluation of individual cells. Similar to in situ hybridization, PCR is among the most sensitive methods available for detection of genomic EBV DNA. In addition to confirming EBV infection and showing the clonality, a ladder of smaller terminal digestion fragments, if present, from linear genomes indicates active EBV replication.
The major immediate-early (MIE) promoter of cytomegalovirus (CMV) controls production of the immediate-early gene products. The majority of commercially available assays for CMV IgG antibodies use either viral lysate preparations or semipurified viral proteins as antibody binding targets; the sensitivities of these assays do not vary widely. Virus-specific T-cell-modulating activity by both types of dendritic cells (DCs) can be regulated through binding of natural ligands, such as lipopolysaccharides and CpG oligodeoxynucleotides, to tolllike receptors on the DCs. Circulating levels of anti-gB antibody have been shown to be inversely proportional to systemic viral load in human immunodeficiency virus (HIV)-infected patients, and high titers of glycoprotein-specific antibodies correlate with the absence of viral DNA in the blood of bone marrow transplant recipients. This suggests a role for antiglycoprotein antibodies in the prevention of CMV disease and in the modulation of its progression. Preliminary trials with one such mixture have shown promising results, but as many as four doses may be required to generate persistent neutralizing antibody responses. Importantly, seronegative recipients of seropositive kidneys who received this vaccine not only developed humoral and cellular immunity but also were protected from severe CMV disease. Adoptive immunotherapy has been proposed as an alternative to prophylactic treatments with antiviral drugs for the prevention of CMV disease in transplant recipients.
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. Monoclonal antibodies suitable for the direct detection of HHV-6 antigens by immunofluorescence have been developed and employed with tissue samples. HHV-6 can be isolated from peripheral blood mononuclear cells (PBMC) grown in primary culture or by cocultivation of these cells with stimulated cord blood cells (CBC) or donor PBMC. A number of tests have been developed for the serodiagnosis of HHV-6 infection, of which indirect immunofluorescence assay (IFA), anti-complement immunofluorescence (ACIF), enzyme immunoassay (EIA), and the neutralization (NT) test have been most commonly employed. The IFA is the most widely used method for detecting. Western immunoblots and radioimmunoprecipitation are other serologic assays that are used to measure antibody to HHV-6. These assays have been used mainly to identify and analyze the role of specific proteins in the immune response to HHV-6. The diagnosis of HHV-6 infection is increasingly being made by the amplification of viral DNA by PCR. The methods for laboratory diagnosis of HHV-7 are essentially identical to those for diagnosis of HHV-6 and currently include the direct examination of clinical specimens for viral antigens by immunohistochemistry or viral nucleic acids by in situ hybridization and PCR antibodies to HHV-6.
Until recently, papillomaviruses and polyomaviruses were grouped together as papovaviruses, but they are now classified as two distinct families. Human papillomaviruses (HPVs) are strictly epitheliotropic viruses that infect squamous epithelia of the skin and mucous membranes. The expression of viral genes is tightly linked to the stages of cellular differentiation. The cervical squamous intraepithelial lesions (SIL) that may result from HPV infections can be detected in Pap smear screening programs and treated effectively. Epidermodysplasia verruciformis (EV) is often familial. Clinically the warts are flat or are in the form of reddish brown macular plaques. Immunization of humans by intramuscular injection with VLPs results in a robust antibody response, with antibody titers far exceeding those resulting from natural infections. A positive test indicates productive infection with any of the HPVs. The infecting HPV type cannot be specifically diagnosed by the test because type-specific antisera are not available. In humans, human polyomavirus JC virus (JCV) is the etiologic agent for the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), while BK virus (BKV) infection is associated with urinary tract infections and nephropathy. Potent antiretroviral drugs have effectively reduced plasma HIV viral loads and increased CD4 cell counts, which could potentially lead to a decreased susceptibility to opportunistic infections such as PML. The antigen responsible for the hemagglutinating activity is located on the Vp1 capsid protein. Diagnostic testing at present relies heavily on DNA-based technologies such as in situ DNA hybridization and quantitative PCR.
This chapter concentrates on serologic methods for diagnosis of human adenoviruses (HAds) infections and recent advances in molecular detection of viral nucleic acid. The most widely used methods for measuring HAd crossreactive, genus-specific antibodies of both the IgG and the IgM classes are enzyme immunoassay (EIA) and indirect immunofluorescence (IIF) assay (also called indirect fluorescent antibody assay). Complement fixation (CF), once used extensively, is the most standardized serologic test for diagnosis of HAd infection. A growing number of reports have linked HAds and acute myocarditis, with and without pericarditis, in infants and young children; sudden infant death associated with myocarditis; and idiopathic left ventricular dysfunction in adults. EIA is now one of the most automated tests in the clinical laboratory and, for the most part, uses the 96-well microplate format, automated plate washers, and readers that define experimental parameters and interpret results. The commercial conjugates for EIA that are currently available are antihuman immunoglobulins labeled with one of two types of enzyme, either horseradish peroxidase or alkaline phosphatase. Detection of viral nucleic acid in clinical materials is the most rapidly expanding diagnostic methodology in medicine, overtaking culture as the “gold standard” for diagnosis of infectious disease. The possibility of PCR contamination must also be evaluated. As with interpretation of serological test results, it is better to have the corroboration of additional methods of diagnosis if possible.
Parvovirus B19 is the smallest (18 to 26 nm in diameter) DNA virus known to infect humans. The clinical spectrum of parvovirus B19 infection may be classified by common and uncommon manifestations. The majority of the children have the hallmark rash characterized by bright red ‘’slapped cheeks’’. The rash may also appear on the torso and extremities. Parvovirus B19 has also been associated with acute fulminant liver failure, myocarditis, mononucleosis-like syndrome, Koplik’s spots, and pneumonia. The indirect immunofluorescence assay was successfully applied to the cellular localization of B19 antigen in tissue and cell culture samples but has not found wide clinical application. The first system developed consisted of tests using native virus from viremic patients as an antigen source. Several groups have synthesized peptides based on published B19 DNA sequences for use as antigen in immunoassays. PCR-based technologies offer exquisite sensitivity and the ability to detect B19 DNA in different types of clinical specimens. Many investigators have now reported the use of PCR to detect B19 in fetal and adult tissues, body fluids, blood products, and cell cultures. Parvovirus B19 infection is widespread in the community. Immunocompromised patients who lack neutralizing anti-B19 antibodies and who present with manifestations of persistent B19 infection benefit from commercial intravenous immunoglobulin. Specific antiviral chemotherapy for parvovirus B19 has not been demonstrated.
Influenza viruses replicate predominantly in the ciliated columnar epithelium of the respiratory tract. Early in infection, large amounts of virus are shed into respiratory secretions which when expelled through sneezing and coughing can be transmitted to close contacts. This chapter focuses on diagnosis of influenza A and B viruses since these types are the major human pathogens. Vaccination is the primary means to reduce the impact of influenza viruses and the disease they cause. Identification of influenza viruses must be done by hemadsorption, hemagglutinin (HA) titration, or fluoescent antibody (FA) staining. Reverse transcriptase PCR (RT-PCR) can be used for the detection of influenza viruses in original respiratory samples from patients, or for the characterization of viruses grown in tissue culture or embryonated eggs. The gold standard for serodiagnosis of infection or response to vaccination with human influenza viruses is the hemagglutination inhibition (HAI) assay. The microneutralization test is an alternative method for the detection of strain-specific antibodies to influenza viruses and in some cases may be more sensitive than the HAI test. The microneutralization test is a highly sensitive assay applicable to the identification of virus-specific antibody in human sera. The neutralization test has several additional advantages for detecting antibody to influenza virus. However, specialized laboratories must continue to use more sophisticated techniques such as virus isolation, RT-PCR, and serological methods in order to monitor the antigenic and genetic characteristics of influenza viruses circulating worldwide.
Respiratory syncytial virus (RSV), human metapneumovirus (HMPV), and the parainfluenza viruses (PIVs) are the major viral causes of acute respiratory tract disease in infants and children. A number of major approaches to the laboratory diagnosis of these virus infections are available: (i) tissue culture for virus isolation from respiratory secretions of the upper and lower respiratory tract, (ii) rapid detection of viral antigen in respiratory secretions or middle ear effusions (in otitis media), (iii) viral RNA detection in respiratory secretions, and (iv) determination of specific antibody responses by serological assays. Diagnostic kits based on enzyme linked immunosorbent assay (ELISA) procedures contain all reagents and provide “self-contained” reasonably sensitive and specific assays for routine diagnosis in the hospital laboratory. ELISA, hemagglutination inhibition (HI), immunofluoresence (IF), neutralization (NT), and Western blot (WB) procedures provide highly specific and especially reliable means for the determination of antibody to RSV, HMPV, and PIV. Selection of amplification primers aims at the sequences of the conserved regions of these genes and is based on analyses of these known sequences from several strains of the virus as available in published databases such as GenBank. For the measurement of the level of antibodies to the four PIVs, individual tests must be carried out with the specific virus type as the test virus in the NT test. Since PIVs do not usually produce cytopathic effect (CPE) in cell culture but do exhibit hemadsorption, the end point of the NT procedure is the demonstration of hemadsorption inhibition (HadI).
Although both measles virus and mumps virus can be isolated in cell culture, isolation may be difficult due to the need to collect specimens very early in the infection process and to the slow proliferation and weak to absent production of cytopathogenic effect by these viruses in traditional tube cell cultures. Newer modified cell culture techniques involving detection of viral antigen in shell vial cultures have improved the sensitivity and speed of measles and mumps virus isolation. Molecular techniques, which do not rely on the presence of viable virus, can be used to detect viral RNA directly in clinical samples. The serologic approach, relying on detection of antibodies for confirmation of infection, has long been the most accessible and reliable diagnostic tool for confirming measles and mumps virus infections. The most widely used methods in measles and/or mumps antibody detection are enzyme immunoassay (EIA) and indirect immunofluorescence assay (IFA). Complement fixation (CF), hemagglutination inhibition (HI), neutralization (NT), and plaque reduction neutralization (PRN) are seldom used in diagnostic laboratories but may be available at reference or research facilities. A brief description of each of these methods is presented in this chapter. Molecular methods are more expensive than virus isolation but may provide a viable approach for confirming the presence of measles or mumps virus. The presence of measles virus RNA in clinical samples is evidence for current or very recent infection or vaccination.
Rubella virus is antigenically stable, and antigenic variation has so far not been an issue for vaccination or serological diagnosis; the significance of the possible emergence of new international subgenotypes of rubella virus is unknown. Reinfection with the virus can occur, but it is almost always asymptomatic and can be detected by a rise in immunoglobulin g (IgG) antibodies. One approach is to demonstrate rubella virus-specific IgM antibody in the infant's serum, which would be diagnostic of congenital rubella. Since the original description of the hemagglutination inhibition (HI) test for rubella, several modifications have been introduced. Commercial enzyme immunoassay (EIAs) are available for testing whole sera for rubella virus-specific IgM. Few laboratories have the techniques or expertise to culture rubella virus, and when virus detection is clinically important, laboratories may want to consider detection of rubella virus RNA by PCR. The challenge viruses most commonly used for rubella virus isolation in AGMK cells are echovirus 11 and coxsackievirus A9. The presence of rubella virus is indicated by little or no cytopathic effects (CPE) in the inoculated tubes and complete destruction of the control cells infected with challenge virus in the absence of rubella virus. An indirect immunofluorescence staining method has also been shown to be specific and sensitive for identifying rubella virus isolates in these cells. The lack of serological responses to rubella virus vaccine in women who do not have detectable antibodies is often due to low levels of neutralizing antibodies.
The enteroviruses are extremely stable in the environment. They retain viability indefinitely at 70№C and for years at 20№C and are not inactivated by ether or chloroform. Infectivity is lost when they are heated at 50№C for 2 min or more. Virions are protected from heat inactivation by addition of divalent cations. UV light, formaldehyde treatment, or sodium hypochlorite (0.3 to 0.5 ppm of chlorine) treatment inactivates infectivity. Heating or UV light converts the infectious N particle to the noninfectious H form. A more frequent but less severe disease is aseptic meningitis, for which enteroviruses are the major cause. Monoclonal antibodies (MAbs) which react with most or all enteroviruses (group-reactive MAbs) may be used in indirect immunofluorescence assays for confirmation. Due to a variety of factors associated with serotyping, including antigenic variation and drift, viral aggregation, expense, and the time required to complete the characterization, there is a great interest and need for development and application of molecular methods for typing enteroviruses. On the basis of molecular and biologic data, these untypeable enteroviruses are now proposed as new enterovirus serotypes. Nucleic acid amplification by PCR is sensitive and specific and is the method of choice for direct detection of enteroviruses. The use of molecular methods, particularly PCR, is an important advance that will contribute greatly to more rapid and sensitive diagnostics for enteroviruses.
The more recently introduced molecular tests that detect and quantify viral genomes complement the information obtained immunologically and, due to rapid turnaround times and the availability of user-friendly commercial kits, have acquired a key position in both the diagnosis and the follow-up of viral hepatitis. Direct detection of the infectious agent has an ancillary role in the laboratory diagnosis of hepatitis A due to the reliability and ease of anti-hepatitis A virus (HAV) immunoglobulin M (IgM) testing. Viral RNA is tested for mainly by reverse transcriptase PCR (RT-PCR). Certainly more important for laboratory diagnosis and disease management, however, are the so-called hepatitis B e antigen (HBeAg)-minus core or precore mutants of the virus, which are unable to express HBeAg due to a stop codon in the C gene, and the drug-resistant variants that are commonly found in patients undergoing therapy. Qualitative nucleic acid-based tests are most useful in assessing the safety of blood donations but prove useful also when a suspicion of active infection based on serological grounds needs to be confirmed. In this case, it is advisable to retest negative patients several times at intervals of several weeks in order to identify the ones who might have intermittent viremia, as is frequently observed in the course of spontaneous resolution or antiviral therapy.
Enzyme immunoassay (EIA), in particular, has been used extensively to establish the incidence and prevalence of rotavirus infection and to define antigenic characteristics of the virus. Antiserum prepared against virus of one serotype tends to neutralize homotypic rotaviruses more efficiently than it neutralizes heterotypic viruses. P1A and P1B are associated with the most virulent human rotaviruses that cause diarrhea. The protocol for the EIA to determine the VP4 serotype is similar to that for the VP7 serotyping EIA described, except that VP4 serotyping antibodies are used. Human infections are most commonly caused by GI and GII noroviruses, representing 15 or more genotypes. By nature of the infection, detection of norovirus nucleic acid is performed with stool samples which may contain inhibitors to RT-PCR. Significant sequence diversity among the multitude of norovirus strains has made it difficult to design primers that detect all strains of norovirus. The RT-PCR methods used most extensively to date are neither the simplest nor the fastest. In addition, verifying the specificity of the amplicon necessitates additional analysis, including probe hybridization and variations of this technique, as well as sequencing the product. Two fluorescence techniques, in single-tube formats, are increasingly studied to detect noroviruses in clinical samples, water, and shellfish: SYBR green dye incorporation into amplicons and the TaqMan fluorogenic detection system. Developing the optimal molecular detection assay for the highly diverse noroviruses continues to be a challenge that is being pursued by scientists throughout the world.
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. Assays that detect virus-elicited immunoglobulin M (IgM) are useful because they detect antibodies produced within days of a primary viral infection. IgG enzyme-linked immunosorbent assay (ELISA) has replaced the hemagglutination inhibition (HI) test for the diagnosis of some infections because the procedure is less cumbersome and titration curves for the two procedures are similar. The indirect immunofluorescence (IF) test for antibodies to most arboviruses is as specific as or less specific than the HI test. The major advantage of both the HI and the complement fixation (CF) test is that species-specific positive-control reagents are not necessary. The serum neutralization test is the most virus-specific test for serologic diagnosis and is used to confirm other serologic testing results. A variety of nucleic acid amplification platforms have been successfully utilized for the detection of arboviruses, including standard reverse transcriptase PCR (RT-PCR), real-time RT-PCR using fluorescent probes, and nucleic acid sequence-based amplification (NASBA). Each of these technologies is described; however, several important issues common to each approach are presented first. The isolation and identification of an arbovirus or its antigen or viral genomic sequences in a clinical sample generally are specific evidence of a recent infection. Three of these arboviruses (VEE, EEE, and WEE viruses) have been classified as possible biological threats. This designation has renewed interest in rapid and sensitive diagnosis of alphaviruses.
The genus Hantavirus is composed of plus-strand RNA viruses within the family Bunyaviridae. The genus is named for the prototypical virus species, Hantaan virus (HTNV). The disease caused by Hantaan virus, now called hemorrhagic fever with renal syndrome (HFRS), came to the attention of western medicine during the Korean war, when soldiers presented with symptoms of fever, hemorrhage, hypotension, and renal failure. Several viruses were isolated that were serologically distinct from HTNV but grouped with Seoul virus. In the early 1980s, the prototypical indigenous North American hantavirus, Prospect Hill virus (PHV), was isolated from the lung tissue of a vole, Microtus Pennsylvanicus. The nucleotide sequence of the amplimer obtained from the lung tissue of a deer mouse captured at a case-patient’s residence was nearly identical to that of the amplimer obtained from the tissues of the patient obtained at necropsy, lending further support to the hypothesis that the patient had contracted the virus from a deer mouse. The existence of a second open reading frame is useful diagnostically because its consistent presence in Sin Nombre virus (SNV) imposes an additional constraint on sequence variability in that part of the genome.
The need for sensitive, specific viral diagnostics and for having procedures in place to handle such agents is essential for the correct identification and containment of outbreaks of viral hemorrhagic fevers (VHFs). Since the procedures for initial isolation, clinical management, and virologic diagnoses of patients with suspected arenavirus and filovirus infections are similar, these taxonomically distinct viruses are discussed together in this chapter. All arenaviruses are maintained in nature by establishing chronic viremia in their reservoir host (almost exclusively rodents) and are transmitted to humans through contaminated animal excreta or occasionally bites. Personto-person transmission (secondary infection) also occurs with Lassa virus (LAS) and less frequently with the South American VHF-causing arenaviruses. Laboratory diagnosis can be achieved in two ways, measurement of host-specific immune responses to the infection and detection of viral antigen and/or genomic RNA. Because of the presence of high titers of infectious virus in blood and tissues even during early stages, antigen and nucleic acid detection plays the most important role in early detection of VHF infections caused by arenaviruses and filoviruses. The method of choice for isolation of pathogenic filoviruses and arenaviruses is the inoculation of appropriate cell cultures, usually Vero cells (especially clone E6), MA-104 cells, or SW-13 cells, followed by examination of inoculated cells at intervals for the presence of viral antigens by using an immunological staining method such as direct immunofluorescence assay (DFA) or immunoperoxidase assays.
Rabies virus, the type species of the genus Lyssavirus of the family Rhabdoviridae, order Mononegavirales, is the causative agent of rabies, an invariably lethal neurological disease of a wide variety of animals and humans. The existence of cryptic rabies and proof that transmission of the disease between humans can occur by organ transplantation indicate that screening of potential donors with encephalitis of unknown origin for rabies is prudent. This can be accomplished by the analysis of tissue biopsies for either rabies virus antigens or nucleic acids and serology, as many victims of clinical rabies exhibit high serum or cerebrospinal fluid (CSF) titers of rabies virus-specific antibodies prior to their death. The dependence of the direct fluorescent-antibody (DFA) test on fluorescence microscopy has led to the development of several enzyme-linked immunosorbent assay (ELISA)-based methods for the detection and quantification of rabies virus antigens that may be more suited for use in the field. The assessment of rabies virus-specific neutralizing antibody titers by fluorescent focus inhibition tests like the rapid fluorescent focus inhibition test (RFFIT) requires technical expertise. Immunization is recommended for individuals whose occupations or other activities may predispose them to exposure to rabies virus. It is noteworthy in this regard that antigenic cross-reactivity between rabies virus G and human immunodeficiency virus type 1 (HIV-1) gp120 has been described, and sera from rabies-immune individuals have occasionally cross-reacted against HIV-1 gp120 in ELISA.
Human T-cell leukemia virus type 1 (HTLV-1) was reported in 1980 by Bernard Poiesz and Robert Gallo as the first retrovirus shown to be pathogenic to humans. As can be surmised from the description of some mechanisms of pathogenesis, the diseases associated with human T-cell leukemia virus (HTLV) infection have inflammatory and/or proliferative attributes. HTLV-1 and HTLV-2 diseases are usually classified as malignant or nonmalignant clinical presentations. Diagnosis of uveitis is based on clinical presentation and the presence of antibodies to HTLV-1 as well as the confirmation of proviral DNA by PCR. Sjögren’s syndrome is an autoimmune disorder complete with the expression of anticentromere antibodies, while the antibodies expressed with sicca syndrome are to HTLV-1. Skin lesions constitute the most common initial clinical presentation of disease and can appear as papules, nodules, infiltrated plaques, tumors, or erythroderma. The treatment of adult T-cell leukemia/lymphoma (ATL) consists of chemotherapy, alpha interferon, and zidovudine. The main cause of death is disease progression combined with hypercalcemia and septicemia. The detection of HTLV-1 and -2 relies on the presence of serum or plasma antibodies and proviral DNA. Enzyme-linked immunosorbent assays (ELISAs), Western immunoblots, and radioimmunoprecipitation assays (RIPAs) are used to confirm repeatedly reactive or inconclusive findings. The interpretation of PCR-restriction fragment length polymorphism findings is based on the comparison to the banding patterns of known standards. HTLV-1- and HTLV-2-seropositive patients should be counseled about prevention of transmission. Patients are advised to use condoms during sexual intercourse and are prohibited from donating blood or blood products.
Coronavirus, (CoV) a genus within the family Coronaviridae, contains a number of enveloped viruses that infect both animals and humans. CoVs have been identified in mice, rats, chickens, turkeys, swine, dogs, cats, rabbits, horses, cattle, and humans and cause a variety of severe diseases, including gastroenteritis and respiratory tract diseases. Severe Acute Respiratory Syndrome (SARS) first appeared as a potentially fatal cause of pneumonia in Guangdong Province of China in November 2002. The first human case was identified on 16 November, and within 6 months SARS spread to 29 countries, infecting 8,098 people and killing 774 individuals. The major route of transmission in humans is droplet infection, aerosolization, and fomites. Deposition of droplets onto the respiratory epithelium probably initiates infection. Whether infection can occur through the oral or conjunctival epithelium remains unknown, but SARS-CoV has been detected in tears. Virus can also be detected in serum, plasma, and peripheral blood leukocytes by RT-PCR; however, the viremia may be short-lived. Isolation of SARS-CoV in cell culture followed by determination of its partial genome sequence led to the first generation of RT-PCR assays for diagnosing SARS. Real-time PCR assays that provide viral loads may also be useful for identifying patients at increased risk for worse outcomes in terms of survival and requirement for intensive care and assisted ventilation.
Poxviruses are large double-stranded DNA viruses that have a wide range of susceptible host species. Monkeypox is of increased interest since its importation into the United States in 2003 and has increased concerns of zoonotic transmission of poxviruses. This chapter addresses molecular and immunological diagnostic issues for detection of viral infections associated with four genera of the Chordopoxvirinae that cause human disease and their public health significance. Diagnostic methods are similar for these viruses and are addressed cumulatively. Parapoxviruses are structurally distinct from other Chordopoxvirinae and commonly cause agricultural disease of sheep, goats, and cattle that may be transmitted by direct contact to humans. The basis for diagnosis of any poxviruses can be attributed historically to smallpox. Detection of humoral antibody responses by serology is an indirect approach to diagnosis and has been a hallmark for laboratory diagnosis of viral infections. The most pragmatic serology tests are reviewed and include hemagglutination inhibition (HI), plaque reduction neutralization testing (PRNT), and enzyme-linked immunosorbent assay (ELISA), with methods described for PRNT and ELISA.
The natural forms of prion disease are shown in this chapter. Diagnostics for transmissible spongiform encephalopathies (TSEs) have become important following the realization that several of them may infect humans (or should be assumed to do so) and that, because of modern agricultural and medicinal techniques, large numbers of people may be put at risk. Also, prophylactic agents and progress in potential methods of treatment for Creutzfeldt-Jakob disease (CJD) patients suggest that diagnostics may be necessary to permit the therapeutics to be used early in a symptomatic phase. Several methods are claimed to show tubulofilamentous particles present in brain that are specific to TSEs and start to appear in the tissues at around half the incubation period of the disease. Standard staining methods can be used for the demonstration of specific histopathological changes in the brain, e.g., hematoxylin and eosin stains. The growth of academic groups in the field of prion research has led to a tendency for each of them to be large enough to declare that their own methods are adequate. The development of many diagnostic systems and methods has meant that many groups have difficulty knowing which would be of most value for individual patients despite the appearance of national and international guidelines. Currently over 20 million cattle are being tested annually for bovine spongiform encephalopathy (BSE) in Europe, and blood transfusion risks may demand that a similar number of blood donations also be tested for the foreseeable future.
This is the introductory chapter to the section Human Immunodeficiency Virus(HIV). The laboratory evaluation of patients with HIV infection has evolved from basic flow cytometry to detailed molecular analysis of viral quasispecies and quantitation of tetramer-positive T cells. As the number of HIV infections worldwide continues to increase, the need for laboratory capabilities for the monitoring of these patients has increased as well. This need is even greater given recent advances in treatment strategies that will hopefully turn this disease into a chronic manageable illness. This section deals with the laboratory immunologic and virologic perspectives of HIV. From determining a patient’s relative risk of developing an opportunistic infection to monitoring immunologic responses to candidate vaccines, the clinical immunology lab plays a major role in the area of HIV. This section deals with the laboratory immunologic and virologic perspectives. The chapter provides brief insights into each chapter under this section.
An important early impetus for the development and testing of serologic methods to detect human immunodeficiency virus (HIV) infection was the need to guarantee the safety of the blood supply. A quick review of the statistics provided in this chapter makes clear the need for the continued development of serologic methods, including point-of-care testing, that are useful especially in resource-poor settings. The standard screening tests for HIV infection involve detection of HIV-specific antibodies. The enzyme-linked immunosorbent assay (ELISA) is the standard screening test for HIV-1 infection. Individuals with indeterminate or positive ELISA results should undergo confirmation testing (generally by Western blotting [immunoblotting]) to determine if the reactivity is primarily directed to HIV-1 infection or secondarily directed to cross-reacting antibodies. It has now become the standard practice in blood banks to screen donated blood samples with one of the new HIV-1-HIV-2 combination ELISAs. The major core protein of HIV-1 is p24. The assay to detect the p24 antigen has been used for many years in the prognostic staging and management of infected individuals and is licensed in the United States as part of routine blood donor screening. Serologic assays to detect antibodies to HIV are the most widely used means of laboratory diagnosis of HIV infection. Most HIV testing algorithms include an ELISA as the screening component and Western blotting as the confirmatory method.
This chapter provides proven methods for the routine immunologic monitoring of the cellular immune status of patients with human immunodeficiency virus (HIV) infection. Since a majority of immunologic assays are performed with peripheral blood mononuclear cells (PBMCs), procedures for the isolation and cryopreservation of PBMCs are also discussed in the chapter. The section describing each procedure is prefaced with an explanation of the rationale behind the described assay, quality control measures necessary for standardization of testing, and practical advice on pitfalls to avoid. When monitoring the immunologic status of patients with HIV infection, isolation of lymphoid cells is often required for performing several immunologic procedures, such as lymphocyte immunophenotyping and measurement of lymphocyte function. Three- and four-color panels of monoclonal antibodies that may be used for the basic monitoring of patients with AIDS have also been discussed. In summary, immunophenotyping of patients with HIV infection can provide the most clinically relevant information as to the immunologic status of these patients.
In the current environment of human immunodeficiency virus type 1 (HIV-1) treatments under highly active antiretroviral therapy (HAART), not only an increase in the number of CD4+ T lymphocytes but also a decrease in the level of plasma viremia is expected; thus, it is essential to be able to quickly and accurately assess viral load. Continuous low-level viral replication, noncompliance issues, and gradual deterioration of the immune system have all contributed to the emergence of drug-resistant HIV-1. HIV drug resistance presents another challenge for clinicians. Until recently, the accepted approach to treating infected individuals with drug-resistant HIV-1 was thorough treatment history and viral load data. RNA is extracted by using guanidium thiocyanate and isopropanol. The use of an internal quantitation standard (IQS) monitors the efficiency of extraction and amplification and eliminates the need for a standard curve. The internal standard and the target sequence compete equally for primer binding and amplification in the PCR. Variables such as the efficiency of amplification and the number of cycles will have the same effect on both templates. The genotypic assays provide sequencing information that can be used to identify known resistance codons. Virus is concentrated from patient plasma by high-speed centrifugation, the virus pellet is lysed and viral RNA is purified using standard procedures.
While the ultimate mechanism of human immunodeficiency virus (HIV) pathogenesis remains elusive, one fortunate consequence of two decades of HIV research has been the development and optimization of new tools to investigate the human immune response, both in health and in disease. A T-cell receptor (TCR) that binds with too much or too little avidity for a self-peptide presented within a self-major histocompatibility complex molecule will cause the death of that T cell. This chapter outlines both old and new tools used in evaluating patients with HIV disease. RNA extractions should be performed in a PCR hood, making sure to run the UV light for sufficient time before beginning in order to avoid any cross-contamination. Two methods, chemical (phenol) and nonchemical (column), are available to isolate RNA from cells. A section of the chapter outlines two popular methods to detect cytokine-secreting antigen-specific T cells using multiparameter flow cytometry. The ability of T cells to rapidly secrete cytokines following stimulation by their cognate antigen is an important hallmark of the memory T-cell response. HLA tetramer technology revolutionized the investigation of antigen-specific T-cell clones. Unlike other assays and technologies, HLA tetramers can enumerate T-cell clones regardless of their functional behavior, such as proliferation, killing, or cytokine production. The ability of lymphocytes to expand exponentially after encountering their cognate antigen is a critical attribute that allows the acquired immune response to contain otherwise lethal pathogenic challenges.
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). The type of pathogen and the location of the infection may give valuable insight into the nature of the immunologic defect. Individuals with complement deficiencies most often present with bacteremia, septic arthritis, and meningitis, caused by encapsulated bacteria. Phagocytic disorders are characterized by bacterial and fungal infections of the skin and abscesses in the reticuloendothelial system (lymph nodes, spleen, and liver). Autoimmune and inflammatory diseases are more commonly seen in particular primary immunodeficiency diseases, most notably common variable immunodeficiency, selective immunoglobulin A (IgA) deficiency, chronic mucocutaneous candidiasis, and deficiencies of early components of the classical complement pathway (C1 to C4). Although immune system dysfunction can be suspected by the clinician after careful review of the history and physical exam, specific diagnoses are rarely evident without the use of the laboratory. However, the types of infections and other symptoms should help to focus the laboratory workup on specific compartments of the immune system. Finally, whenever primary immunodeficiency is suspected, consideration must also be given to secondary causes of immunodeficiency, including human immunodeficiency virus infection, therapy with anti-inflammatory medications (e.g., corticosteroids), and other underlying illnesses (e.g., lymphoreticular neoplasms and viral infections, such as infectious mononucleosis).
Central to the adaptive immune system, T cells protect the host from intracellular pathogens by mediating cytolytic activity and releasing Th1 cytokines. In addition, through release of soluble mediations such as interleukin 4 (IL-4) and IL-10 and interactions with antigen-presenting cells and B cells, T cells regulate the production of antibody against protein antigens. Nevertheless, a profound selective deficiency in the T-cell lineage such as in CD3δ deficiency is sufficient to produce a full phenotype of severe combined immunodeficiency (SCID) with extreme susceptibility to microbes of even low pathogenicity. Genetic and immunological features of SCID have been discussed in this chapter. However, a significant proportion of SCID patients has normal or near normal number of circulating lymphocytes (Omenn’s syndrome, major histocompatibility complex class II deficiency, and ZAP-70 deficiency). Flow cytometry analysis will help decipher these cases and will aid in pinpointing the molecular defect by providing insight into the number of B cells and NK cells. It is therefore recommended that all infants with a putative diagnosis of SCID have their lymphocyte subsets analyzed. Finally, since the genes responsible for many forms of SCID have already been identified, it is important to perform mutation analysis.
This chapter discusses antigen-receptor signal transduction pathways for both T cells and B cells and describes methods to evaluate these pathways in children with inherited immune deficiencies. ZAP-70 deficiency was the first described T-cell receptor (TCR)-associated protein tyrosine kinases (PTKs) defect in humans. Moreover, intracellular cytokine expression using fluorescence-activated cell sorter (FACS) analysis may be useful in characterizing the scope of T-cell defects. Fluorimetric methods require more cells but do not require concomitant cell staining with potentially stimulatory MAbs. This assay is best done using fresh peripheral blood mononuclear cells (PBMC), but bone marrow-derived lymphocytes, B-cell lines (BCL), or T-cell lines (TCL) rested overnight in medium without IL-2 or mitogen may also be used. Distal TCR signal transduction events, as well as the interleukin 2 (IL-2) pathway, may be analyzed by measurement of IL-2 production in response to TCR-mediated stimuli. In vitro T-cell proliferative studies are useful in the screening of immunodeficient patients for proximal signal transduction defects. TCL and BCL may be the only source of patient cells; therefore, analysis of signal transduction defects using transformed lines may be complicated by the effects of HTLV-1 and EBV, and possibly HVS, on lymphocyte signaling pathways. The described methods in this chapter are valuable both to screen and to diagnose signaling defects in patients with inherited T- or B-cell immune deficiencies. Signaling pathways resulting in abnormal T- or B-cell activation and development of immune deficiency are the focus of much investigation at present.
In recent years several rare autosomal recessive disorders that result in antibody deficiency have been reported. Some antibody deficiencies are part of a more broadly expressed systemic disorder or part of an immunodeficiency that affects T cells and/or NK cells as well as B cells. The possibility of immunodeficiency should be considered in any patient who is hospitalized for a major infection requiring intravenous therapy. Most patients with X-linked agammaglobulinemia (XLA) develop recurrent or persistent infections in the first 4 to 8 months of life, and the majority are recognized to have immunodeficiency at less than 3 years of age. Defects in μ heavy chain account for about one-third of the patients with autosomal recessive agammaglobulinemia. Mutation detection is the most practical way of making a definitive diagnosis. Single gene defects of the immune system may have features in common with common variable immunodeficiency (CVID). Recent studies suggest that analysis of B-cell phenotype may be useful in identifying patients with CVID who have more severe disease. Either whole blood or density gradient-separated mononuclear cells can be used to examine lymphocyte cell surface markers. A variety of techniques can be used to provide mutation detection. The advantages and disadvantages of some techniques are listed in this chapter. A detailed history of past infections, a careful family history, and a physical examination that focuses on sites typically involved in antibody deficiencies should come before any laboratory tests.
Complement comprises an interactive system of more than 30 plasma and cell membrane-associated recognition molecules, enzymes, cofactors, control proteins, and receptors. It plays an important role in the host’s response to infection by coating microbial surfaces with complement fragments that enhance uptake and killing by phagocytes. As a major effector arm of the innate immune system, complement serves as a link between many of the activities of acquired immunity and other defense mechanisms, with ties to diverse cell signal responses in an ever-increasing number of tissues. The classical pathway (CP) is critical for the clearance of immune complexes, and it also participates in the removal of apoptotic cells. The phenotype of C1q and other early CP component deficiencies is closely linked to diseases in which these processes are impaired, such as systemic lupus erythematosus (SLE), and rheumatologic disorders such as anaphylactoid purpura, vasculitis, and membranoproliferative glomerulonephritis. The primary synthesis of C1q is by cells of monocyte/macrophage lineage, with follicular dendritic cells as a secondary site. C1q deficiency is an autosomal recessive condition, with deficient patients having little or no detectable protein or C1q function in the circulation. Recombinant or monoclonal antibody complement inhibitors have been used in ischemia-reperfusion injury, cardiopulmonary bypass surgery, and autoimmune disorders. Soluble CR1 acts against the C3 and C5 convertases of both the alternative pathway (AP) and CP, and a humanized anti-C5 monoclonal antibody that prevents cleavage of C5 to C5a and C5b has been effective in animal models and clinical trials.
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. There are three basic mechanisms by which neutropenia can occur: decreased neutrophil production, increased neutrophil destruction, or abnormal neutrophil trafficking (either defective release of neutrophils from the bone marrow or an abnormal increase in the marginated or tissue pools). Recently, an autosomal dominant disorder with mild neutropenia due to neutrophil retention in the bone marrow (myelokathexis) has been molecularly characterized. The determination of a qualitative defect of neutrophil function is more complex than the simple determination of neutropenia, since it requires the demonstration of a dysfunctional phenotype. The major classes of neutrophil defects highlight the major neutrophil functions: adhesion, chemotaxis, phagocytosis, degranulation, and killing. Nitroblue tetrazolium (NBT) reduction and dihydrorhodamine (DHR) oxidation are the simplest tests available and are discussed. Chemiluminescence and staphylococcal killing are also discussed since they not only are used in the demonstration of CGD functional defects but also occasionally may help in diagnosis. Despite the important role of myeloperoxidase (MPO) in the neutrophil, clinical disease from MPO deficiency is quite rare and has been reported mostly for diabetics with disseminated Candida infections. MPO is an important marker of myeloid maturation, appearing at the promyelocyte stage of development.
This is the introductory chapter to the section Allergic Diseases. This section covers the important topics of the allergen, in vivo and in vitro confirmatory IgE antibody testing, mediators and markers of allergic inflammation, and application of the analytical measurements to food allergy and hypereosinophilic syndromes. This chapter presents brief insights into each chapter discussed in the section. Chapter 105, by deVore and Slater, examines assays that are used in the quantification and standardization of allergens that are glycoproteins which elicit allergic reactions in susceptible individuals. Chapter 106, by Damin and Peebles, examines in vivo confirmatory assays for the detection of allergen-specific IgE antibody in patients suspected of having allergic disease. Chapter 107, by Hamilton, examines the diagnostic analytes that are measured in the diagnostic allergy clinical and research laboratory to aid in the diagnosis and management of human allergic disease. Chapter 108, by Schroeder and Saini, overviews mediators and markers of inflammation that are associated with immediate-type hypersensitivity reactions. Chapter 109, by Fleischer and Wood, discusses diagnostic tests that are useful in the workup of food allergy. In chapter 110, Klion overviews the clinical features, pathogenesis, and variants of hypereosinophilic syndromes.
This chapter talks about natural or recombinant proteins or glycoproteins used in the diagnosis and/or treatment of immunoglobulin E (IgE)-mediated allergic disease. The purpose of allergen standardization is to ensure that the extracts are well characterized in terms of allergen content and that variation between lots is minimized even among different manufacturers. In the United States, the use of a biological model of allergen standardization has permitted the assignment of bioequivalent allergen units (BAU) for most standardized allergens. Although skin testing is an essential component of the allergen standardization program, it is not intended for routine use in the testing of manufactured lots of extracts prior to release. For the Hymenoptera venom allergens, the potency determination is also based on the content of the known principal allergens within the extract, hyaluronidase and phospholipase, which is determined by enzyme activity. Individual allergens may be measured and detected by various approaches using monospecific antisera or antibodies. Assay designs using these antisera include the radial immunodiffusion (RID) assay, crossed immunoelectrophoresis/ crossed radioimmunoelectrophoresis (CIE/CRIE), and enzyme-linked immunosorbent assay (ELISA) variants (direct, two-site, and competition). Each of the assays utilizes monospecific antisera or antibodies to detect and quantify the specified allergen. The identity of an allergen extract may be verified by visualizing the separated allergen proteins on the basis of their size and isoelectric points.
Prick-puncture and intradermal skin testing are the most commonly used methods for the diagnosis of immediate hypersensitivity in allergic disease, while airway challenges may be used in the evaluation of rhinitis and asthma. This chapter describes the technique and utility of in vivo skin testing, intranasal allergen challenge, and specific and nonspecific lower airway challenge testing. These tools provide clinically useful information in the evaluation of the atopic patient. Allergen skin testing is considered to be the most convenient, least expensive, and most specific screening method in the diagnosis of allergic diseases. General principles that relate to in vivo examination are discussed in this chapter. Several methods have been developed to test the effect of antigen exposure on the nasal mucosal surface in allergic reactions. First, a specific antigen is identified by a combination of history consistent of allergic symptoms upon exposure to the antigen and positive skin testing, followed by intranasal challenge with that antigen. The two most commonly used methods of intranasal challenge include instillation of antigen into the nares either by spray pump or by nebulizer. The contraindications for methacholine and histamine challenges are essentially the same as for whole-lung antigen challenge. Persons exposed to oxidizing pollutants, smokers, and cystic fibrosis patients may also have increased reactivity based on methacholine and histamine challenges. Challenge procedures can be extremely useful in the investigation of allergy.
This chapter overviews clinically used and research methods for the quantification of total and allergen-specific IgE antibodies and allergen-specific IgG antibodies. The performance of immunoassays for these analytes has continued to improve with the availability of new solid-phase matrices, conjugate labeling technology, standardized reference reagents, and data processing methods. One monoclonal antibody-based noncompetitive solid-phase two-site immunoenzymetric assay (IEMA) is described here as the procedure of choice for research investigations of total serum IgE. Because total serum IgE is a regulated analyte as defined by the amended Clinical Laboratory Improvement Act of 1988, licensed clinical laboratories providing this analytical measurement must demonstrate proficiency in an external proficiency survey such as the Diagnostic Allergy (SE) survey conducted by the College of American Pathologists. A modification of the total serum IEMA described above allows the quantification of the level of "free" or non-omalizumab-bound IgE in serum. A 1992 study highlighted that venom-specific IgG levels of <3.5 μg/ml are associated with an increased risk of allergic reactions upon a resting during the first 4 years of immunotherapy with yellow jacket or mixed vespid venoms. In summary, the diagnostic allergy laboratory uses a number of immunoassay methods to measure IgE and IgG antibodies that aid the clinician in the diagnosis and management of individuals with allergic disease. As more is learned about genetic polymorphisms that predispose individuals to allergic disease, molecular biology methods may also be employed in the diagnostic allergy laboratory of the future.
With the discovery of immunoglobulin E (IgE) as the source of reaginic activity in serum, the release of histamine from leukocyte suspensions challenged with a specific antigen has been used as a reliable in vitro correlate of immediate hypersensitivity. Although histamine remains the most commonly measured mediator released following this IgE-mediated reaction, it is now well established that other mediators are also released from basophils and are important markers of allergic inflammation. This chapter highlights the growing interest in the use of flow cytometry assays to monitor activation-linked markers (e.g., CD63, CD69, and CD203c) on basophils and the applicability of such assays as surrogate indicators of mediator release. The radioenzymatic assay (REA) has the advantage of detecting very low levels of histamine, with a sensitivity of approximately 10 pg/ml. The REA is not subject to interference from high protein levels, which makes it an excellent protocol for measuring plasma histamine levels. Measurement of the release of preformed histamine from peripheral blood basophils challenged with a specific antigen is among several tests available, and histamine release remains a valuable in vitro correlate of immediate hypersensitivity reactions. Mediators such as LTC4 and IL-4, are generated by basophils upon IgE-dependent activation, and in vitro assays have recently been developed to measure these products. These assays, combined with those available for the detection of several other mediators occurring in biological fluids, have produced data that have significantly added to our understanding of the parameters, mechanisms, and pharmacologic control of allergic inflammation.
Food allergy is defined as an adverse immunological response to food. Some of the tests discussed in this chapter not only aid in the diagnosis of food allergies, but also are useful in monitoring the natural history of patients' food allergies over time, from diagnosis to oral tolerance. Once food allergy has been identified as the likely cause of symptoms, confirmation of the diagnosis and identification of the implicated food(s) can begin. There are a number of tools that aid in the diagnosis of food allergy, some of which are more commonly used, and they vary in their ability to provide an accurate diagnosis. Available studies include in vivo tests such as skin prick and intradermal testing, oral food challenges (OFCs), elimination diets, and patch testing and in vitro tests such as quantification of food-specific IgE and basophil histamine release (BHR). Tests that have not proven helpful in food allergy diagnosis include quantification of food-specific IgG, total serum IgE levels, BHR, and serum tryptase concentrations. Tests that need further study but show some promise include the atopy patch tests (APT), which may aid in the diagnosis of non-IgE-mediated disorders and delayed reactions to foods. Finally, important features of the studies mentioned in this chapter for the described tests that need to be appreciated by clinicians are (i) that the data generated may be particular to the study population and test material and (ii) that the age and clinical disease of the patients are important variables.
The term hypereosinophilic syndromes (HES) was first used in 1968 by Hardy and Anderson to describe three patients with marked peripheral eosinophilia, hepatosplenomegaly and cardiac and/or pulmonary symptoms. Additional subgroups of HES with distinctive clinical manifestations await characterization at the molecular and immunologic levels. The first step in the diagnosis of HES is to exclude other disorders associated with marked peripheral eosinophilia. These include parasitic infections, drug hypersensitivity reactions, neoplasms, and immunodysregulatory disorders associated with secondary eosinophilia. If routine laboratory results (including human immunodeficiency virus (HIV) testing) and diagnostic testing do not lead to an alternative diagnosis, computed tomography of the chest, abdomen, and pelvis, as well as a bone marrow examination, should be performed in all patients to exclude occult malignancy before a diagnosis of HES is confirmed. Other diagnostic tests that may be helpful in establishing the diagnosis include the measurement of thymus and activationregulated chemokine (TARC) levels in serum and the assessment of the lymphocyte production of eosinophilopoietic cytokines. As the number of chemotherapeutic agents with specific molecular and immunologic targets continues to grow, the resolution of these issues will become increasingly important for the appropriate management of patients with primary eosinophilic disorders.
This is the introductory chapter to the section Systemic Autoimmune Diseases. Systemic autoimmune diseases are often associated with the production of autoantibodies that recognize a diverse array of cytoplasmic and nuclear antigens. The detection of disease-specific autoantibodies in asymptomatic individuals may permit early diagnosis and preventative treatment. Good examples are antimitochondrial antibodies in primary biliary cirrhosis, anti-Sm, RNP, Ro(SSA), and double-stranded DNA autoantibodies in systemic lupus erythematosus (SLE), and specific autoantibodies associated with rheumatoid arthritis, polymyositis, and scleroderma, all of which may appear prior to the onset of clinical manifestations. The chapters in this section reflect both the evolving technology and the need for reliable confirmatory tests, which frequently are older or more labor-intensive assays that are not readily available. Chapters also deal with the mainstream autoantibody assays employing solid-phase antigens, both natural and recombinant. They discuss the clinical use of antineutrophil cytoplasmic antibody testing in the diagnosis of vasculitis syndromes and the newer tests that detect autoantibodies against specific neutrophil proteins.
This chapter reviews the history and the method for antinuclear autoantibodies (ANAs) testing on HEp-2 cells and its utility in a laboratory setting. Antinuclear antibody tests have their origin in the “L.E.” cell phenomenon, which was the observation that neutrophils from patients with systemic lupus erythematosus (SLE) ingested other leukocytes. Widespread commercial production of HEp-2 cells and the development of national quality control schemes ensured that the test entered worldwide routine laboratory usage. The nuclear envelope is the membrane that maintains the integrity of the nucleoplasm during interphase. Important autoantibodies are found against most of the cell cycle-related structures that have been mentioned, such as the centromeres, proliferating cell nuclear antigen, mitotic spindle proteins, and centriole proteins. The pattern on HEp-2 cells is usually sufficient to identify most of these autoantibodies. The majority of laboratories use a non-affinity-purified, fluorescein-conjugated second antibody directed against IgG, -A, and -M heavy chains (or anti-IgG heavy and light chains). The HEp-2 cells should be observed by using an epifluorescence microscope fitted with filters appropriate for fluorescein detection. The largest variation in results reported to quality control schemes relates to differences in fluorescence intensity of the samples. The ANA immunofluorescent HEp-2 test is intended to be used for screening and titration of circulating antinuclear antibodies. Autoantibodies occur in both physiological and pathological conditions.
Antinuclear antibodies (ANA) are markers for systemic autoimmune disease but also can be seen at low titers in healthy individuals. Thus, there has been considerable interest in identifying ANA subsets with greater diagnostic specificity for particular diseases. Autoantibodies specific for systemic lupus erythematosus (SLE), systemic sclerosis (SSc), polymyositis or dermatomyositis (PM/DM), and other systemic autoimmune disorders have been identified and are of considerable importance as diagnostic markers. The prevalence of some of the more useful autoantibody markers for various diseases is summarized. Double immunodiffusion (DID) (Ouchterlony method) is a classic immunoassay to detect interactions of antigens and antibodies. Antigenic proteins or protein complexes in the cell extract and antibodies in the serum diffuse toward one another in the gel and form insoluble antigen-antibody complexes when the antigen-to-antibody ratio is near equivalence. Gel diffusion and enzyme-linked immunosorbent assay (ELISA), unlike Western blot assays, allow the detection of autoantibodies to native anti-genic determinants, but the composition of the antigenic particles and fine specificities of the autoantibodies cannot readily be determined. Immunoprecipitation is a highly sensitive and specific technique for detecting a variety of autoantibodies. Immunoprecipitation is the gold standard for detecting autoantibodies against Ku/DNA-dependent protein kinase (DNA-PK). Autoantibodies against aminoacyl tRNA synthetases are diagnostic markers for PM/DM and the antisynthetase autoantibody syndrome.
This chapter focuses on the methodology in purification of recombinant autoantigens used in the authors' laboratories and recent advances in multiplex immunoassays that are suitable for autoantibody detection. Key aspects related to the production of recombinant autoantigens are discussed briefly in this chapter. The construction of recombinant proteins to be used in the detection of autoantibodies should take into consideration the suitability of the construct design and the ease of purification of resulting proteins. Each assay has limitations in terms of determining the sensitivity, specificity, and positive predictive value for the detection of autoantibodies. The chapter focuses on addressable laser bead technology and recent experience with it in a clinical laboratory setting with addressable laser bead immunoassays (ALBIA). In a clinical service laboratory setting an autoantigen panel based on ALBIA was evaluated and compared to the routine diagnostic protocol, which analyzed 870 sequential unselected sera received over a 6-month period. In this study, the sera were submitted primarily by clinicians who were considering a diagnosis of systemic rheumatic disease. Unselected sera from various disease cohorts were also studied to determine if the frequency of autoantibodies as detected by the ALBIA was consistent with the published frequency of autoantibodies. Taken together, there are advantages and challenges that come with the adoption of the ALBA platform to detect autoantibodies. There are now several ALBIA kits on the market that detect eight or more autoantibodies relevant to systemic rheumatic diseases.
Antibodies to DNA (anti-DNA) are prototypic autoantibodies found prominently in the sera of patients with systemic lupus erythematosus (SLE). While DNAs from various species likely differ in antigenic structure, anti-DNA antibodies in SLE sera appear to bind epitopes expressed widely on DNA, independent of its origin. Although expressed pathologically, anti-DNA antibodies nevertheless differ in their ability to mediate inflammation and tissue damage, in particular glomerulonephritis. While the role of anti-DNA in immune complex renal disease may underlie the correlation with disease activity, anti-DNA antibodies may have other functions that contribute to this association. Most clinical laboratories now perform tests that specifically detect anti-dsDNA antibodies, although anti-ssDNA can provide useful information for assessing disease activity; anti-ssDNA assays are also easier to perform. Currently, the methods available for anti-DNA include the radioimmunoassay (RIA), the Crithidia luciliae immunofluorescence (CLIF) assay, and the enzyme-linked immunosorbent assay (ELISA). The flurometric PicoGreen assay is similar to the Farr assay in principle but has the advantage of not using radiolabeled DNA. The new technology of microarrays allows the detection of several antibodies in a single experiment.
With new developments in the therapy of rheumatoid arthritis (RA) and evidence that therapy is most effective when started early in the disease process, it becomes increasingly important to develop criteria that will permit the accurate diagnosis of disease prior to the onset of radiographic changes. Rheumatoid factor (RF) is an antibody directed against the Fc portion of human IgG. The presence of RF in the blood is one of the diagnostic criteria used for RA. First, there may be an RA-specific overexpression of citrullinated antigens in the synovium, leading to an immune response. Second, the presence of citrullinated proteins is perhaps a common phenomenon in any inflamed (synovial) tissue but RA patients show an abnormal humoral response to them. Anti-cyclin citrullinated peptide (anti-CCP) antibody levels can change substantially in RA patients; most show elevated levels at the first visit, followed by a decrease in the majority of cases and fluctuation over time. It also has been hypothesized that the fluctuation in the anti-CCP levels may simply reflect the natural history of the autoantibody production over a course of time and that the level may peak early in the disease process, irrespective of therapeutic interventions, before declining and reaching baseline. However, more recent studies suggest that by evaluating the presence of either autoantibody (RF or anti-CCP), sensitivity for RA is increased without substantially altering the specificity.
Of the several types of antiphospholipid antibodies (aPL) described, lupus anticoagulant (LA) antibodies and anticardiolipin antibodies (aCL) are the types most clearly associated with antiphospholipid antibody syndrome (APS), the latter being the focus of this chapter. The Venereal Disease Research Laboratory (VDRL) and rapid plasma reagin tests were then developed, both employing purified cardiolipin combined with cholesterol and lecithin as the antigen. Classification of patients with APS considers both clinical and laboratory criteria. The syndrome is classified as primary APS when not associated with another disease and accounts for over 50% of cases. The first category of aPL, LA antibodies, are detected with certain phospholipid-dependent coagulation assays. Definite APS is present when at least one clinical criterion and one laboratory criterion are present. LAs are antibodies that are prothrombotic in vivo but display anticoagulant properties in vitro (i.e., they prolong certain coagulation tests). Antibodies associated with APS that are detected in aCL ELISAs were originally thought to bind to the phospholipid molecule. An appreciation of the presence of aPL in systemic lupus erythematosus (SLE) patients, and their association with thrombosis, led to the development of an RIA for detection of aCL antibodies and subsequently an ELISA-based format. As discussed in the chapter, cutoff levels should be validated in each laboratory performing aPL testing. Alternative phospholipids and/or cofactor antibodies may be detected in patients with APS. Occasional patients with APS do not have aCL, LA, or β2GPI antibodies.
This chapter concentrates on vasculitides which are associated with presence of antineutrophil cytoplasm antibodies (ANCAs), it focuses on idiopathic smallvessel vasculitides (SVV), and mentions some of the clinical conditions where other types of neutrophil-specific autoantibodies (NSA) are commonly produced. The diagnosis of a primary vasculitic condition must rest on sound clinical judgment of more or less characteristic constellations of symptoms and features, with some being indicative of vasculitis and others reflecting just a general inflammatory condition (arthralgias, fever, fatigue, loss of appetite, hypersedimentation). Although the chapter focuses on the methodologies used to detect ANCAs, it also stresses important aspects of setting clinically validated assay cutoff values, controlling laboratory performance quality, knowing frequent pitfalls, and how to exercise troubleshooting. It also mentions possibilities for reporting laboratory data with due consideration toward differential diagnostics, potential clinical utility, and interpretation. Monoclonal mouse antibodies to proteinase 3 (PR3), myeloperoxidase (MPO), and elastase (EL) have been used to capture the respective antigens from cell extracts, and the captured antigen has then been used as the target in an enzyme-linked immunosorbent assay (ELISA).
This is the introductory chapter to the section Organ-Localized Autoimmune Diseases. Autoimmunity is common, while autoimmune disease is not. Autoimmune disease can be divided into two types. The first type - the systemic diseases - are conditions in which the whole body may be involved. The second category consists of the organ-specific autoimmunities. More and more of the assays are now based on enzyme-linked immunosorbent assay using purified antigens or recombinant antigens. Certain new assays use immunoblots of tissue extracts or digested purified antigens. These assays have promise for screening several antigens or epitopes simultaneously. The role of the clinical laboratory is changing from one in which new assays are developed to one of evaluating the new technologies.
This chapter describes the various procedures currently used to detect circulating autoantibodies in patients with endocrine disease, i.e., thryoiditis, Graves’ disease, insulin-dependent diabetes mellitus, Addison’s disease, and pernicious anemia. The general method of indirect immunofluorescence (IF) is the screening test used most frequently to detect tissue- or organ-specific autoantibodies. Commercial hemagglutination kits for thyroglobulin antibodies are less sensitive than the tanned-cell hemagglutination (TCH) test described. Indirect IF performed on sections of human or monkey thyroid can demonstrate antibodies to thyroglobulin, CA2, and microsomes of thyroid epithelial cells, and antinuclear antibodies. The most commonly used tests for determining thyroid peroxidase (TPO) antibodies are indirect IF and commercially available hemagglutination assays or ELISAs. Separate diabetes mellitus categories consist of gestational diabetes and ‘’specific types of diabetes’’ usually with defined mutations or accompanying pathology (e.g., pancreatic abnormalities, endocrinopathies, or drug induced). The current insulin autoantibodies (IAA) assay is not able to distinguish between natural IAA and induced insulin antibodies. The current major format for determination of islet autoantibodies uses fluid-phase radioimmunobinding assays, applied to all three major islet autoantibody tests, including glutamic acid decarboxylase (GAD) autoantibodies (GAA), ICA512AA, and IAA. The majority of sera from type 1 diabetes patients target more than two epitopes on ICA512 molecules, while non-disease-related antibodies usually react with only one epitope. Anti-islet autoantibodies find their primary application in the differential diagnosis of the forms of diabetes and in predicting risk of progression to overt diabetes.
Antiglycolipid antibodies are viewed as forming an important part of the natural autoantibody repertoire directed towards microbial carbohydrate structures, which has the potential to be expanded in both specific and nonspecific ways. This may explain their ubiquitous presence in both healthy and diseased patient populations and may account for some of the misleading studies that have claimed disease-specific associations, further confounded by technical difficulties in their measurement. This chapter only addresses their relationship to autoimmune neuropathy, for which diagnostic testing is widespread. In chronic peripheral neuropathy syndromes, antiganglioside antibodies are usually IgM, occurring either as paraproteins (often monoclonal gammopathies of undetermined significance) or as polyclonal antibodies. Antiganglioside autoantibodies are referred to by their specificity, either in terms of individual gangliosides (e.g., anti-GM1 IgG antibodies) or in terms of the reactive carbohydrate epitope (e.g., anti-Gal(β1-3)GalNAc IgM antibodies). When generalizing, it is often more appropriate to use the term antiglycolipid antibodies (as opposed to antiganglioside antibodies), since many neuropathy-associated autoantibodies react with glycolipids which are not strictly gangliosides, such as sulfated glucuronyl paragloboside (SGPG), sulfatide, asialo-GM1, and galactocerebroside, as these molecules do not contain sialic acid.
This chapter focuses on the detection of antimitochondrial autoantibodies (AMA) in primary biliary cirrhosis (PBC) and the detection of liver-kidney microsomal antibodies (LKM antibodies) in autoimmune hepatitis (AIH). PBC is an autoimmune disease of the liver characterized by autoimmunity-mediated destruction of intrahepatic bile ducts with progressive inflammatory scarring and, eventually, liver function failure. Immunologically, PBC is characterized by the presence of AMA in the circulation and the infiltration of T cells into the liver. AMA can be detected by the following methods: (i) indirect immunofluorescence (IF), (ii) immunoblotting, and (iii) enzyme-linked immunosorbent assays (ELISAs). LKM antibodies have been defined due to their typical immunofluorescent staining of liver and kidney tissue. LKM autoantibodies can be detected by the following methods: (i) IF, (ii) competitive ELISA, (iii) immunoblotting with human liver microsomes, and (iv) immunoblotting with recombinant antigens.
The dermis and epidermis have a dense network of adhesion proteins and fibrous structural proteins that provide mechanical strength to the skin. A number of these adhesion molecules are known to become targets of autoantibodies, and antigen-antibody interactions in the skin lead to the clinical manifestations of autoimmune bullous skin diseases. Demonstration of the characteristic autoantibodies in the tissue and circulating in the blood is the critical diagnostic feature of numerous such diseases. In most of these diseases, the circulating autoantibodies have also been shown, by passive transfer in animal models, to directly cause the characteristic tissue injury. For each of these diseases, this chapter describes the expected findings in tissue and serum and outlines immunochemical tests that may be useful in diagnosis. There are four major forms of pemphigus: pemphigus vulgaris (PV), pemphigus foliaceus (PF), paraneoplastic pemphigus (PNP), and IgA pemphigus. All forms are characterized by a loss of normal epidermal cell-to-cell adhesion (acantholysis) and by the presence of pathogenic autoantibodies reacting against desmosomal adhesion molecules. Pemphigus is a potentially lethal disease; to establish the diagnosis with certainty, the presence of both tissue-bound and circulating autoantibodies must be demonstrated.
Cardiovascular diseases comprise a broad spectrum of disorders which cause cardiac dysfunction. Vascular diseases such as hypertension, atherosclerosis, and acute and chronic coronary artery disease, which adversely influence systemic hemodynamics, indirectly cause cardiac dysfunction by causing acute myocardial infarction (AMI) or by promoting heart muscle disease. Cardiomyopathies are classified as hypertropic, restrictive, dilated, arrhythmogenic right ventricular, unclassified, or specific. Specific cardiomyopathies describe heart muscle diseases that have been further defined based in part on their etiologies and their association with specific systemic or cardiac disorders. This chapter focuses exclusively on the current knowledge of immunologic aspects involved in the etiology and pathogenesis of cardiomyopathies and the application of immunological and molecular techniques for measuring and characterizing relevant biomarkers used in the detection and assessment of myocardial damage and in determining the diagnosis and prognosis of related cardiovascular diseases.
Several diseases of the kidney with overlapping histological and clinical features are characterized by a rapid loss of renal function, crescentic glomerulonephritis, and sometimes lung hemorrhage. Serological assays for the detection of anti-neutrophil cytoplasmic antibodies (ANCA) and anti-glomerular basement membrane (anti-GBM) autoantibodies are important diagnostic tools in the evaluation of patients with rapidly progressive renal failure and/or pulmonary hemorrhage, providing a rapid noninvasive method for establishing a diagnosis. These tests are sensitive and highly specific and can provide definitive diagnostic information for the effective clinical management of patients with a rapidly progressive course of disease. This chapter describes the detection of circulating anti-GBM antibodies by serological techniques, including Western blot analysis and enzyme-linked immunosorbent assay (ELISA).
Experimental studies indicate that infertility may have an immune basis, resulting from spontaneous or induced autoimmune disease that targets the testis, the ovary, or the spermatozoa. This chapter describes spermatozoal antibody-mediated infertility, with emphasis on the methods of sperm antibody detection with respect to the laboratory procedure, clinical indications, and interpretation of results. It focuses an emerging assay for detection of antibodies to ovarian antigens. It also discusses the human testicular and ovarian autoimmune diseases and the diagnostic dilemma they present. Autoimmunity ensues following stimulation by foreign microbial antigen that mimics gonadal antigen, when antigen is presented to T cells in the context of a vasectomy-induced granuloma, or invoked by endogenous antigen when an immunoregulatory mechanism, such as regulatory T cells, is defective. Spermatozoal antibodies may be detected in serum, or they are bound to the ejaculated spermatozoa. An important indication of the presence of antisperm antibody is an abnormal postcoital test (PCT), particularly when the semen quality is normal and the cervical mucus, obtained at late follicular phase, is well hydrated and acellular. While an abnormal PCT is suggestive of the presence of antibodies, a negative result does not preclude antibody testing. Patients with infertility for which there is not an apparent cause, despite rigorous clinical evaluation, are also candidates for antisperm antibody testing. Ovarian dysfunction is determined by abnormalities in menstrual cycle patterns and measurement of early follicularphase follicle-stimulating hormone and estradiol.
The diagnostic usefulness of serological testing for celiac disease (CD) is well established since the antigens and the immune response involved in its pathogenesis are now known. Serological tests for inflammatory bowel diseases (IBDs) (Crohn’s disease and ulcerative colitis [UC]) have been relatively recently developed, and although clinical use is growing, controversy about their role in diagnosis and management continues. CD is now recognized to be one of the most common inherited diseases in humans, and serological testing has become an indispensable tool for detecting it. Individuals with CD make an immune response directed at a combinatorial antigen involving gliadin (the ethanol-soluble protein of gluten) and tissue transglutaminase (tTG), a ubiquitous enzyme which uses gliadin as a source of glutamine residues that it adds to tissue proteins. Antigliadin antibody (AGA) was the first serological test used to detect CD and to monitor patient compliance with a gluten-free diet. Enzyme immunoassay (EIA) is the method most commonly used to measure AGA. Inflammation in Crohn’s disease is segmental, with affected areas alternating with normal areas in the small intestine. Treatment of Crohn’s disease was formerly limited to anti-inflammatory drugs, rest, and good supportive care. Within the past few years, a new approach to therapy has proven very successful in patients with predominantly small intestinal involvement. Monoclonal antibodies to tumor necrosis factor alpha (TNF-α), a cytokine critical in the inflammatory pathways in Crohn’s disease, or TNF-α receptor analogues are able to block TNF-α activity and dramatically reduce inflammation and clinical symptoms.
A chronic form of autoimmune thrombocytopenia exists and is the clinical course most often seen in adults. With characterization of the immunopathological features of autoimmune thrombocytopenia, a variety of methods have been developed to measure the antiplatelet autoantibodies that mediate the immune destruction of platelets in this disorder. The risk of chronicity increases with increased age of the child; otherwise, there are no prognostic features, either clinical or laboratory, that will predict whether a child with acute immune thrombocytopenia will recover spontaneously. The pathogenic factor present in the plasma reacted with normal and autologous platelets and could be isolated and removed from the gamma fraction of plasma by adsorption with platelets. Patients with chronic autoimmune thrombocytopenia typically present with petechiae and mucosa bleeding. Symptoms may be present for months, or some patients may experience more acute manifestations. Quantitative measurements based on antiglobulin consumption give falsely high values for platelet-associated immunoglobulin because antiglobulin binds differently to membrane-bound immunoglobulin G (IgG) than to the IgG in the solution used to calibrate the standard curve.
This chapter deals with the identification of antiretinal immune reactivity in patients with retinal diseases. An extraordinary feature of the eye is its immune status. The immune response that occurs within the eye is different from the systemic immune response, and this unique characteristic has been referred to as immune privilege. Retinal autoimmunity exists as a naturally occurring disease in humans and as an experimentally designed animal model system. Sympathetic ophthalmia (SO) is an ocular inflammatory (autoimmune) disease that occurs after a perforating injury to one eye. Cancer-associated retinopathy (CAR) is most commonly associated with small-cell carcinoma of the lung, but it has also been reported for patients with breast, endometrial, and other cancers. Posterior ocular onchocerciasis is characterized by atrophy of the retinal pigment epithelium (RPE), and as lesions advance, subretinal fibrosis occurs. Toxoplasmosis, which occurs in over 500 million humans worldwide, is caused by the obligate intracellular parasite Toxoplasma gondii. T. gondii is also the most frequently identified infectious agent in posterior uveitis, and Toxoplasma retinochoroiditis is an important cause of blindness in young adults. Measurement of antiretinal immune reactivity is difficult to perform in the clinical laboratory. Analysis of immunocytochemical or immunofluorescent staining of retina tissue sections requires special training for interpretation.
This is the introductory chapter to the section Cancer. Proven and investigational tumor markers are mostly proteins that have proven or anticipated use in the diagnosis and management of cancer patients and patients receiving immunologic therapies. Diagnostic oncology and immunology laboratories all over the world currently measure a number of biomolecules released from tissue into body fluids that are markers of tumor presence and growth. The measurement of these analytes assists oncologists in making more accurate diagnoses and managing their patients with cancer, especially as it relates to monitoring therapy, detecting cancer recurrence, and assessing the patient’s prognosis. This has been a challenging area of diagnostic immunology because most tumor markers are not elevated under all cancerous conditions and others are elevated under non-cancer conditions such as benign or nonmalignant disease.
This chapter reviews the design, performance, and application of tumor marker assays; interferences; specific tumor markers; and the impact of new technologies on the discovery of novel tumor markers. In most populations, there is an overlap between the noncancer and cancer groups, since individuals without cancer may exhibit elevated levels of a marker and some individuals with cancer may not show elevations in a particular marker. The possible applications for tumor markers include screening, diagnosis, monitoring of therapy, detection of disease recurrence, and determination of prognosis. The prevalence of most cancers in the population is low, but with the inappropriate sensitivity and specificity of some tumor marker assays, false-positive results may be generated. Surgical removal of the tumor should result in a dramatic fall of the tumor marker, consistent with its half-life. Antibodies can be employed for specific targeting of analytes in many complex biological matrices, including blood (whole or a portion thereof), urine, and other fluids. The specificity of the antigen-antibody interaction, coupled with the exquisite sensitivity of enhanced signal detection methods available today, makes immunoassays the method of choice in laboratory medicine.
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 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 and molecular (DNA- or RNA-based) tumor markers. The application of modern immunologic techniques and concepts has permitted the development of a conceptual framework that may be used to decipher the morphological diversity of these neoplasms and has shown the relationship of lymphoid and mononuclear phagocytic neoplasms to the normal immune and hematopoietic systems. In recent years, an increasing number of mouse monoclonal antibodies have become available for use in paraffin sections, eliminating the need to perform immunohistochemistry on frozen tissue sections. Lymphoblastic malignancies (LBL) are neoplasms of precursor T and B lymphocytes. They may present clinically as either lymphoblastic lymphoma or acute lymphocytic leukemia (ALL), and morphologically the T and B-lineage varieties of LBL are indistinguishable. The ability of the cells to form follicles correlates with the presence of dendritic reticulum cells (DRC), which can be identified in frozen and paraffin sections by using monoclonal antibodies reactive with CD21, CD23, CD35, or clusterin. Hodgkin’s Lymphoma (HD) is an unusual neoplastic disorder because the neoplastic cells represent only a minority of the cells present in the tumor mass.
Biologic therapies have gained considerable acceptance in recent years. A broad array of biologic agents have become available for the treatment of inheritable or acquired immunodeficiency, autoimmune diseases, cancer, or persistent infections. Biologic therapies can be categorized as follows: (i) monoclonal antibodies (MAbs), (ii) cytokines, (iii) growth factors, (iv) activated cells, (v) cellular products, (vi) immunotoxins, and (vii) other immunomodulatory agents. Ex vivo preclinical studies of the new agent with human mononuclear cells (MNC) are often helpful in providing a rationale for targeting therapy to a particular subset of these cells or for focusing mechanistic studies on a particular cell population. The rationale for monitoring immunologic parameters in clinical trials with biologic agents is based on the premise that these agents achieve therapeutic effects as a result of their ability to modify one or more components of the patient’s immune system. In clinical trials with cytokines, it is important to measure the pharmacokinetics of the cytokine used for therapy as well as the levels of secondary cytokines in serum, which might be responsible for toxicity of the therapy. Monitoring of immune functions generally requires considerable cell numbers, fairly large volumes of blood or other body fluids, and facilities to process these samples for cell recovery. Cryopreservation may introduce artifacts, even when a rate-controlled drop in temperature is implemented. A vast array of labeled MAbs suitable for multicolor and multiparameter flow analyses are commercially available, allowing the measurement of the proportions of various immune cell subsets.
A new paradigm for cancer diagnostics is that the concept of a biomarker for cancer detection and monitoring is not limited to a single protein but can comprise a proteomic pattern of many individual proteins and the changes this pattern undergoes when tissues transform from a normal to a malignant state. This chapter addresses the issues related to technology development, validation, and quality assurance, and discusses trends in future diagnostic strategies. The importance of mass spectrometry (MS) to the fields of proteomics and cancer diagnostics is undeniable. Although the mass spectrometer can be found in many designs and is used for various functions, nearly all mass spectrometers can be described as the combination of three basic components: the ion source, the mass analyzer, and a detector. The chapter describes the importance of the three most popular ionization techniques for MS: matrix-assisted laser desorption ionization (MALDI), surface-enhanced laser-desorption ionization (SELDI), and electrospray ionization (ESI). ESI MS requires more extensive sample preparation and theoretical expertise than MALDI or SELDI-MS; however, it is the most powerful MS technique available. Diagnostic strategies based on immunoproteomics exploit the natural response of the human immune system by identifying antigens associated with major histocompatibility complex (MHC) class I and class II molecules that are uniquely associated with cancer cells.
Early attempts in human transplantation were limited by the immunosuppressive regimens which comprised primarily steroids, irradiation, and azathioprine. The development of clinical transplantation has fostered growth in understanding of the alloimmune response and of the major histocompatibility complex and in histocompatibility assessment. For much of the past 50 years of transplantation, the primary assay for histocompatibility testing, which can be used both for typing and for alloantibody detection, has been the complement-dependent cytotoxicity test. Molecular typing methods and solid-phase immunoassays are enabling histocompatibility laboratories to adapt testing to meet the changing needs of transplant patients. Three of the chapters in this section focus on the methods that are being used to monitor and evaluate transplant patients. For transplants with some degree of HLA mismatching, accurate determination of humoral sensitization is required to assess the risk of engraftment failure due to antibody-mediated rejection. The remaining chapters in this section address these newer issues for bone marrow and stem cell transplantation.
Molecular biology techniques aimed at identification of HLA polymorphism at the gene level have largely replaced HLA typing assays based on identification of HLA proteins. The HLA genes encode at least six different HLA molecules, HLA-A, -B, and -C (class I molecules) and HLA-DR, -DQ, and -DP (class II molecules). The HLA molecules are encoded by the most polymorphic genetic loci known in humans. Each HLA allele is designated by the name of the gene followed by an asterisk and a four- to eight-digit number indicating the allele. Different HLA alleles defined by DNA typing can specify HLA proteins which are indistinguishable by methods based on protein identification, such as serology. DNA from HLA-characterized reference cells is usually derived from Epstein-Barr virus-transformed B lymphoblastoid cell lines. Molecular biology-based HLA typing methods utilize DNA as a starting material. PCR is used to generate a large number of copies of an HLA gene for rapid detection of HLA types. PCR uses sequence-specific primer pairs (a 5’ [sense] primer and a 3’ [antisense] primer) and Taq polymerase to specifically amplify selected segments of DNA. DNA is negatively charged due to its phosphate composition; thus, amplified DNA will move toward the positively charged pole in an agarose gel matrix. Amplification is used to obtain sufficient copies of a specific HLA gene for analysis by DNA sequencing.
This chapter focuses on HLA-specific antibodies. The term donor-specific antibody (DSA) is used to refer to an antibody (ies) specific for donor HLA. The chapter deals with the technical aspects of obtaining the information necessary to evaluate humoral sensitization to HLA antigens in the transplant patient. There are two categories of test used to assess a humoral response, the crossmatch and the antibody screen, the latter of which may be further subdivided into screens that test simply for the presence of antibody and those designed to provide antibody characterization. The advantages and disadvantages of the various techniques for testing antibodies are discussed in the chapter. The major disadvantages of the antibody screen are that it neither tests donor cells nor defines antibody directly. Additional specialized techniques discussed in the chapter may be useful in cases in which it may be crucial to confirm the presence or absence of a particular antibody. The increasing understanding of the role of the humoral response in transplantation indicates the need for certain types of information: (i) does the patient have antibodies; (ii) if so, what are the specificities, titers, and Ig classes; (iii) are the antibodies increasing or decreasing in strength or breadth; (iv) has the patient had antibodies in the past; and (v) has the patient experienced potentially sensitizing events.
Mixed leukocyte culture (MLC) is perhaps the most widely used of cellular assays. MLC has been used clinically for donor selection, predominantly for bone marrow transplantation. In contrast to MLC, which measures a bulk response, limiting dilution assay (LDA) is a quantitative tool. Mycoplasma contains the thymine kinase enzyme, which interferes with nucleic and amino acid metabolism and subsequently with DNA replication in the contaminated cell culture. In solid-organ transplantation, the histological analysis of allograft biopsies remains the "gold standard" for diagnosing rejection. Since the ultimate goal is the early detection of activated cells infiltrating the allograft and not in vitro priming, it also deals with methods that minimize the possibility of priming against the donor antigens in vitro. Several of the assays outlined in this chapter are being implemented in the clinical laboratory. Researchers compared helper and cytotoxic antirecipient T-cell frequencies in recipients who had received unrelated-donor bone marrow. They found that the helper T lymphocytes (HTLp) and cytotoxic T lymphocytes (CTLp) assays provided similar predictive information for outcome. The HTLp method is more rapid and less labor-intensive and, thus, may be more useful for donor selection in unrelated-donor bone marrow transplantation. A recent multicenter study evaluated the Cylex ImmuKnow assay for the measurement of immune response in immunosuppressed transplant recipients. The long-term goals of using the cellular assays described in this chapter are to assess accurately the changing immuno-logical statuses of transplant recipients, predict long-term graft outcome, and provide the immune response information needed to individualize immunosuppression therapy.
Improved knowledge of the alloimmune repertoire, development and clinical application of new therapeutics, advances in surgical techniques, and effective infection prophylaxis have advanced transplantation to the forefront of modern medicine. This chapter provides an outline of the immune cascade contributing to allograft rejection, emphasizes molecular protocols that have been applied to investigate gene expression, and summarizes molecular correlates of rejection of human kidney, heart, lung, liver, or pancreatic islet cell allografts. The advent of an in vitro assay, PCR, to amplify the nucleic acid sequences has greatly reduced the amount of starting material required for the identification and measurement of mRNAs in biological samples. Cytotoxic T lymphocytes (CTL) destroy target cells via multiple effector mechanisms. Cardiac allograft vasculopathy (CAV) is a form of chronic rejection in which intimal thickening leads to accelerated transplant coronary artery disease. Acute rejection of lung allografts is distinguished by perivas-cular mononuclear cell infiltration of venules and arterioles. Chronic rejection of lung allografts is a major diagnostic and therapeutic challenge and a significant contributor to morbidity and mortality. Nucleic acid-based strategies have proven useful for the detection and quantification of gene expression in the clinic. Global expression profiling with technologies such as microarrays will add to our knowledge and provide further insights into molecular pathways, and several pathways are likely to be responsible for the rejection process. The ultimate objective, personalized medicine for the transplant recipient, is an accomplishable goal.
Regulation of natural killer (NK) cytolytic activity is a function of engagement of one or more NK surface receptors that may be activating or inhibitory. Two of the primary sets of receptors present on human NK cells are called the killer immunoglobulin (Ig)-like receptors (KIR; previously known as the killer inhibitory receptors) and the CD94/NKG2 lectin-like receptors. These two receptor gene families and their roles in the immune response are the topic of this chapter. KIR are found primarily on NK cells but also on NKT cells (a subset of memory T cells with NK receptors), where they may modulate the adaptive immune response by competition with the T-cell receptor for ligand or by modulation of the response depending on which KIR genes are present. Because killing by NK cells is dependent on the strength of signal resulting from the inhibitory-activating receptor competition, pathogenesis can result when either the levels of the signals or the ligands are perturbed. Signaling in NK cells occurs through a variety of receptors, the most well known of which is CD16 (Fc gamma RIII). The signaling pathways are dependent upon the family of NK receptor and upon whether the receptor is activating or inhibitory. The involvement of KIR genes in viral infections and potentially in bone marrow transplantation suggests that KIR typing may have wider application in the future. For this reason, KIR typing by molecular methods is becoming standardized through an ongoing external quality control program.
This chapter discusses fundamental aspects of laboratory assessment of hematopoietic chimerism after blood and marrow transplantation. Clinical indications for chimerism testing in blood and marrow transplantation include posttransplant monitoring of engraftment kinetics and stable mixed chimerism as well as detecting relapse and graft loss. Additional applications include detecting cells engrafted from a third party and distinguishing monozygotic and dizygotic twins. Chimerism testing is routinely used after allogeneic blood and marrow transplantation to monitor the status of the allograft and to detect relapse. In the early years of bone marrow transplantation, chimerism testing was primarily limited to use for monitoring donor engraftment. In 2005, the predominant methods for chimerism testing involve amplification of short tandem repeats (STR) loci. There are many situations in which there is coexistence of donor and host hematopoietic cells, and this phenomenon is referred to as mixed chimerism. In patients with mixed chimerism, the proportion (percentage) of donor cells can change, and this condition is described as increasing mixed chimerism if the percentage of donor cells is increasing or decreasing mixed chimerism if the percentage of donor cells is decreasing. For blood and marrow transplantation, chimerism analysis has become an important component of posttransplant monitoring. In addition to the clinical indications, chimerism testing can be used to test for the presence of third-party cells derived from blood transfusions, maternal cell engraftment in immunocompromised children, and maternal cell contamination of umbilical cord blood units.
This chapter provides a broad overview of the major agencies and regulations which have had an impact on laboratory practices. Laboratory personnel must be kept constantly updated by reading appropriate professional articles and reviewing the appropriate Web pages of the responsible agencies and organizations for the latest information. Various governmental agencies, regulations, organizations, etc., are referred to in this chapter. In addition, an extensive list of websites is provided to help the reader obtain further information regarding specific agencies, regulations, and programs. Clinical laboratory improvement amendments (CLIA) has established four categories of testing,: waived, moderate complexity, provider-performed microscopy, and high complexity. The CLIA regulations issued by the federal government have mandated that all laboratories performing clinical testing must undergo regular inspections by an agency or organization with deemed status, e.g., the CAP, JCAHO, or New York State. The process to become accredited by the College of American Pathologists (CAP) begins with the submission of an application. Generally a medical technologist surveyor is assigned to review the laboratory’s activities and policies. A significant ongoing requirement of all accrediting agencies is the regular performance of testing on blinded samples. A number of organizations are involved in issuing credentials or licenses for individuals who demonstrate technical and management skills in clinical immunology. Several professional and voluntary organizations have developed standards for the testing of clinical specimens and provide reagents for the standardization of assays.
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At A Glance
Johns Hopkins Univ., Baltimore, MD. Provides a concise review of basic principles underlying each procedure of laboratory findings. Topics include introduction to protein analysis, clinical indications and applications of serum and urine protein electrophoresis, and more. For immunologists. Previous edition: c2002. DNLM: Immunologic Techniques-Laboratory Manuals.
Description
This three-inch thick heavy hardcover book is the 7th edition of the ASM Manual of Molecular and Clinical Laboratory Immunology. This book used to be known as the Manual of Clinical Laboratory Immunology, but had a name change to emphasize the growing contribution of molecular-based testing to Clinical Laboratory Immunology. There are also new editors (Barbara Derrick, Robert Hamilton, James D. Folds) carrying on the tradition of excellence established by Noel Rose.
Purpose
The purpose of this book is to provide a comprehensive reference for Clinical Immunology Laboratories and as a guide for Directors of Clinical Immunology Laboratories. The authors are world renowned in their areas of expertise, and the editors have done an admirable job of providing the most up to date information and a cohesive text style for this hefty book.
Audience
This book would appeal to Clinical Laboratory Scientists (in training or in practice), especially those working in Clinical Immunology Laboratories. Pathologists are also an obvious audience. Other physicians, mid-level providers, medical students or allied health students with an interest in Clinical Immunology and wanting an in-depth "is all" reference book would want to have this book.
Features
This weighty tome consists of 17 sections, each of which is divided into multiple chapters. Each section covers topics of interest that fit the section grouping. At first glance, it was interesting to see how this "horizontal" text combines areas traditionally separated into "vertical" laboratory disciplines; there are entire sections on Clinical Chemistry, molecular testing, Hematopathology, and Medical Microbiology that in and of themselves would be comprehensive texts on their own. Each chapter contains relevant current information, may include "recipes" for performing specific assays, and insight into interpretation of results. One small minor issue for me was that analytical and clinical sensitivity of various new immunological assays was well discussed, but relatively little was included for analytical and clinical specificity. Another very small issue was the inconsistency of organizational names throughout the text - in particular, NCCLS was referred to in one chapter by its original text name, as
NCCLS in another chapter, and only with its current name (CLSI) in the chapter on Laboratory Management. These few minor issues aside, this book remains the authoritative text for Clinical Immunology.
Assessment
What's to compare? This book *is* the standard for Clinical Immunology textbooks. Make sure your bookshelf and upper body strength is adequate to use this book!
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Reviewer: Valerie Ng, PhD MD (Alameda County Medical Center/Highland Hospital)
Review Date: Unknown
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