Clinics in Laboratory Medicine Current Issue

Direct to Consumer Testing: The Role of Laboratory Medicine

Vinay S. Mahajan

MILENKO JOVAN TANASIJEVIC, MD, MBA

Elsevier

CLINICS IN LABORATORY MEDICINE

Immune-targeted therapeutics are being used in cancer. Immune “checkpoint inhibition” provides promise for prolonged disease-free patient survival. Use of immune checkpoint inhibitors in cancer has coincided with the onset of immune-related adverse events (irAEs). irAEs are caused by a break in host self-tolerance, which can be deadly. Acute management of irAEs is complicated by difficulty making a prompt clinical diagnosis. The goal is to maximize anticancer benefit while minimizing irAE risk. We currently lack diagnostic tools to assess pretreatment irAE risk and facilitate diagnosis. Current immunologic understanding of irAEs is discussed with an emphasis on how patients with congenital syndromes of T-cell activation may inform this understanding. The prospects of improving diagnostics for and treatment of irAEs are discussed.

Antiphospholipid syndrome (APS) is as an autoimmune disease characterized by thrombosis and/or specific pregnancy-related morbidity associated with persistent antiphospholipid antibodies, namely, lupus anticoagulant and IgG and IgM antibodies to cardiolipin and beta2 glycoprotein I. Optimal antibody detection plays a central role in diagnosis and classification. This review discusses antiphospholipid antibodies helpful for diagnosing APS. It includes the criteria and noncriteria antiphospholipid antibodies, methods for their detection, and challenges for clinical reporting and interpretation. The significance of using specific noncriteria antiphospholipid tests in an integrated diagnostic approach with criteria antiphospholipid makers for the diagnosis and management of APS is also reviewed.

The presence of antinuclear antibodies (ANAs), which include autoantibodies to extractable nuclear antigens (ENAs), in the sera of patients with connective tissue diseases provides useful immunologic and pathophysiologic insight into the nature of their disease. This article discusses the most commonly used diagnostic modalities for detecting and quantitating the presence of ANA: indirect immunofluorescence assay, enzyme-linked immunosorbent assay, and multiplex bead technology, which serve as useful screening tests. We also review testing for autoantibodies to ENAs, which are often helpful to confirm the diagnosis of a specific connective tissue disease.

The recent explosion in our knowledge of the molecular basis of immune regulatory mechanisms has paved the way for rapid developments in immunosuppression, immunotherapy, allergy, autoimmunity, and immunodeficiencies, all of which have impacted the practice of clinical laboratory immunology. The advent of immune checkpoint inhibitors and cell therapies in recent years has led to a surge of interest in clinical immunology. Advancements in lasers, microfluidics, and advanced electronics have resulted in the proliferation of robust and affordable flow cytometry or cell-sorting instruments that can withstand the demands of a clinical lab.

For decades, autoantibody detection has comprised the bulk of clinical laboratory immunology. However, most immune disorders are caused by imbalances in both humoral and cellular immunity. Our knowledge of the immune system has grown exponentially, resulting in new treatment paradigms in immunology. Extensive functional characterization of lymphocyte subsets is routinely carried out in a research laboratories, facilitated by the emergence of high-dimensional analysis technologies for low cell numbers. It will not be long before these approaches enter the diagnostic realm. This chapter outlines emerging trends in laboratory immunology testing with a focus on deep immune profiling or high-dimensional testing modalities.

“With the increasing application of biotechnology to the realm of pharmacology and therapeutics, the types of biological treatments available have significantly expanded. Currently, recombinant proteins, humanized antibodies, or rationally engineered monoclonal antibodies are used on a regular basis in the clinical setting. Moreover, cell-based therapeutics with molecularly rewired antigenic specificities are becoming increasingly common in oncology and are actively being developed for a broad range of diseases. Nonetheless, there has been a significant lag between the development of these technologies and the emergence of assays that can monitor these novel interventions.”

The outcomes of kidney transplantation show a steady improvement with an increasing number of transplantations and decreasing incidence of acute rejection episodes. Successful transplantation begins with a comprehensive immunologic risk assessment and judicious choice of therapeutic agents. In this review, we discuss the trends in transplant immunosuppression practices and outcomes in the United States. We discuss practical testing algorithms for clinical decision making in induction therapy and fine-tuning maintenance immunosuppression. We introduce assessment tools for immune monitoring after transplantation and speculate on future directions in management.

Although the gold standard for diagnosis of immunoglobulin E (IgE)-mediated food allergy is an oral food challenge, clinically relevant biomarkers of IgE sensitization, including serum-specific IgE and skin prick testing, can aid in diagnosis. Clinically useful values have been defined for individual foods. More recently, specific IgE to particular protein components has provided additional diagnostic value. In summary, food allergy diagnostics to evaluate IgE sensitization are clinically useful and continue to evolve to improve evaluation of IgE-mediated food allergies.

Laboratory assays of immune cell function are essential for understanding the type and function of immune defects. These assessments should be performed in conjunction with a detailed history and physical examination, which should guide the evaluation of patients with a suspected immune deficiency. Laboratory assays of immune cell function are critical for assessing and demonstrating the functional impact of genetic mutations. Advances in diagnostic techniques continue to expand the ability of clinicians and researchers to understand the complex immune pathophysiology that underlies these disorders.

The complement system is a critical component of both the innate and adaptive immune systems that augments the function of antibodies and phagocytes. Antigen-antibody immune complexes, lectin binding, and accelerated C3 tick-over can activate this well-coordinated and carefully regulated process. The importance of this system is highlighted by the disorders that arise when complement components or regulators are deficient or dysregulated. This article describes the pathways involved in complement activation and function, the regulation of these various pathways, and the interpretation of laboratory testing performed for the diagnosis of diseases of complement deficiency, exuberant complement activation, and complement dysregulation.

The discovery of antineutrophil cytoplasmic antibodies (ANCA) helped establish ANCA-associated vasculitis as a separate and well-defined clinical entity. Its progressive incorporation into the clinical diagnosis algorithms has made ANCA testing a cornerstone immunoassay embedded in the management of ANCA-associated vasculitis. After its description by indirect immunofluorescence, proteinase-3 and myeloperoxidase were identified as principal ANCA targets. ANCA, and proteinase-3 and myeloperoxidase immunoassessment, have undergone iterative rounds of improvement in sensitivity and specificity. This article traces landmarks in the development of ANCA tests, describes common pitfalls arising during ANCA interpretation, and discusses new technologies to improve the future of ANCA testing.

Accurate diagnosis of inflammatory arthritides remains a challenge because of substantial clinical overlap. To achieve a granular classification for informing clinical decisions, numerous potential serologic biomarkers have been identified. Rheumatologists have settled on rheumatoid factor and anti–citrullinated protein antibodies for the diagnosis of rheumatoid arthritis (RA) based on specificity and sensitivity and their ability to be integrated into clinical algorithms. These biomarkers should be interpreted in their specific clinical context. This article discusses the serologic basis for the diagnosis of RA, how these biomarkers have framed conceptualization of the pathogenesis of RA, and the inherent limitations in their use.

Flow cytometry is an incredibly powerful diagnostic tool in the evaluation of primary and secondary immune deficiencies. Assay design and setup involves a methodological consideration of specimen collection, marker and fluorochrome selection, antibody titration, instrumentation, compensation, gating, reference range development, and cross validation. Commonly used analyses for lymphocytes are the lymphocyte subset, T-cell subset, B-cell and T-cell naive/memory, double-negative T-cell, and plasmablast panels. Flow cytometry has direct clinical applicability to the workup of severe forms of primary immune deficiency disorders and is used diagnostically and for therapeutic monitoring in the context of secondary immune deficiency disorders.

Immunology testing is relevant for the diagnosis of many autoimmune conditions. However, diagnostic pitfalls arise owing to incorrect interpretation of results and incomplete understanding of the underlying technique or immune-mediated condition. Here, we review the diagnostic considerations related to commonly used immunology tests. Specifically, we summarize the caveats pertinent to the interpretation of rheumatoid factor, antinuclear antibodies, antiphospholipid antibodies, antineutrophil cytoplasmic antibodies, and serum IgG4 testing.

Primary immunodeficiency diseases are a heterogeneous group of rare inherited disorders of innate or adaptive immune system function. Patients with primary immunodeficiencies typically present with recurrent and severe infections in infancy or young adulthood. More recently, the co-occurrence of autoimmune, benign lymphoproliferative, atopic, and malignant complications has been described. The diagnosis of a primary immunodeficiency disorder requires a thorough assessment of a patient’s underlying immune system function. Historically, this has been accomplished at the time of symptomatic presentation by measuring immunoglobulins, complement components, protective antibody titers, or immune cell counts in the peripheral blood. Although these data can be used to critically assess the degree of immune dysregulation in the patient, this approach fall short in at least 2 regards. First, this assessment often occurs after the patient has suffered life-threatening infectious or autoinflammatory complications. Second, these data fail to uncover an underlying molecular cause of the patient’s primary immune dysfunction, prohibiting the use of molecularly targeted therapeutic interventions. Within the last decade, the field of primary immunodeficiency diagnostics has been revolutionized by 2 major molecular advancements: (1) the onset of newborn screening in 2008, and (2) the onset of next-generation sequencing in 2010. In this article, the techniques of newborn screening and next-generation sequencing are reviewed and their respective impacts on the field of primary immunodeficiency disorders are discussed with a specific emphasis on severe combined immune deficiency and common variable immune deficiency.