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36 Laboratory Diagnostics inAutoimmune Diseases

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Other non CBP-specic ANA, mainly anti-Centromere and anti-SSA/Ro – 52 kDa antibodies, are often detected (10–30%) in CBP patients, conferring, according to some authors, unfavourable prognostic features to the disease course. This nding correlates with the observation of the association of CBP with other autoimmune diseases, such as scleroderma, Sјogren’s syndrome and CREST syndrome.

Finally, it is interesting to report new markers currently under study for the laboratory diagnosis of CBP, namely two new self-antigens (KLHL12 and HK1), genetic markers, particular metabolic proles, miRNAs and epigenetic factors.

With regard to CBP, it is interesting to note, in summary, how the increased knowledge of the serological associations of the disease, together with the widespread use of noninvasive tests, such as laboratory tests, has signicantly modied the initial clinical presentation in recent years, often allowing an early diagnosis to be made with respect to the detection of advanced liver disease.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic syndrome characterized by inammatory brosis of the intra- and extrahepatic bile ducts.

SPC typically affects young males and is commonly associated with inammatory bowel disease, especially ulcerative colitis (CU).

From the point of view of laboratory diagnostics, most patients present an elevation of serum alkaline phosphatase and γGT that may be accompanied by a modest increase in transaminases; unlike CBP, in CSP, the search for AMA antibodies is negative. The determination of anti-neutrophil cytoplasmic antibodies (ANCA) with a pANCA panel in ethanol, an autoantibody marker also present in a high percentage of patients with CU (approximately 80%), was found to be useful for diagnosis.

In summary, laboratory diagnostics of hepatic autoimmune diseases, both in terms of diagnosis differential and early diagnosis, has made important progress in recent years and, without doubt, the prospect is open to new developments. In this eld, an issue that could be of great interest in the near future is the improvement in the standardization of methods and the denition of new diagnostic algorithms.

At present, the detection of specic autoantibody proles is essential, among autoimmune liver diseases, for the diagnosis of AIH and CBP, while the diagnosis of CSP is made by means of the picture provided by endoscopic retrograde cholangiopancreatography. The crucial value of autoantibodies for the differential diagnosis of EAI and CBP is expressed in the consequent therapeutic choices.

Failure to detect autoantibodies does not exclude the presence of an autoimmune hepatopathy, for example CBP AMA negative.

In this diagnostic eld, in perspective, the percentage of patients negative for AMAs, which still represent the key

immunological marker for CBP, will be signicantly reduced by the use of new antigen-specic laboratory tests (ELISA and Immunoblotting tests) based on the use of recombinant antigens.

In addition, the increased use of automated systems that include a full panel of related CBP self-antigens, including the nuclear antigenic targets gp210 and sp100, will contribute to the optimization of new diagnostic algorithms. Conducting multicentre studies evaluating newly diagnosed CBP patients may effectively allow the evaluation of these new analytical tools in the screening and differential diagnostics of CBP.The results of these studies could contribute to redesign the entire diagnostic process, starting from the formulation of a reasonable clinical suspicion to the request for tests, up to the use of the most appropriate autoantibody tests and ensuring through the shared implementation of common algorithms a harmonization among different laboratories.

In other words, clinical governance and standardization in the eld of immunological laboratory diagnostics of autoimmune liver diseases, as for other autoimmune diseases, are two essential elements to enhance the potential of autoantibody biomarkers both in diagnostic and prognostic elds.

Recommended Readings

Accordo, ai sensi dell’articolo 4, del decreto legislativo 28 agosto 1997,

n. 281, tra il Governo, le regioni e le province autonome di Trento e

di Bolzano sul “Protocollo per la diagnosi e il follow up della celia-

chia” (Rep. Atti n. 125/CSR) Gazzetta Ufciale della Repubblica

Italiana Serie generale n. 191 del 19 agosto 2015 Bizzaro N, Pasini P (1999) Correlazione tra titolo e pattern degli anti-

corpi anti-nucleo in immunouorescenza indiretta e positività degli

anticorpi anti-ENA.Med Lab 7:419 Bizzarro N, Wiik A (2004) Appropriateness in the anti-nuclear antibody

testing: from clinical request to strategic laboratory practice. Clin

Exp Rheumatol 22:349–355 Bonaguri C, Melegari A, Ballabio A et al (2011) Italian multicentre

study for application of a diagnostic algorithm in autoantibody

testing for autoimmune rheumatic disease: conclusive results.

Autoimmun Rev 11:1–5 Bonaguri C, Melegari A, Dall’Aglio P etal (2009) An Italian multicen-

tre study for application of a diagnostic algorithm in autoantibody

testing. Ann N Y Acad Sci 1173:124–129 Catassi C, Gatti S, Fasano A (2014) The new epidemiology of celiac

disease. J Pediatr Gastroenterol Nutr 59(Suppl 1):S7–S9 European Association for the Study of the Liver (2017) EASL Clinical

Practice Guidelines: the diagnosis and management of patients with

primary biliary cholangitis. J Hepatol 67(1):145–172 Floreani A, Caroli D, Variola A et al (2011) A 35 year follow-up of

a large cohort of patients with primary biliary cirrhosis seen at a

single centre. Liver Int 31:361–368 Ford AC, Sandborn WJ, Khan KJ etal (2011) Efcacy of biological

therapies in inammatory bowel disease: systematic review and

meta-analysis. Am J Gastroenterol 106:644–659 Gabeta S, Norman GL, Liaskos C etal (2007) Diagnostic relevance and

clinical signicance of the new enhanced performance M2 (MIT3)

500

https://t.me/medicina_free

T. Trenti et al.

ELISA for the detection of IgA and IgG antimitochondrial antibodies in primary biliary cirrhosis. J Clin Immunol 27:378–387

Gatselis NK, Dalekos GN (2016) Molecular diagnostic testing for pri-

mary biliary cholangitis. Expert Rev Mol Diagn 9:1001–1010

Gershwin ME, Makay IR, Sturgess A, Coppel RL (1987) Identication

and specicity of cDNA encoding the 70 kd mitochondrial antigen recognized in primary biliary cirrhosis. J Immunol 138(10):3525–3531

Granito A, Muratori P, Quarneti C etal (2012) Antinuclear antibodies

as ancillary markers in primary biliary cirrhosis. Expert Rev Mol Diagn 12:65–74

Ha C, Mathur J, Kornbluth A (2015) Anti-TNF levels anti-drug antbosi-

dues, immunosuppressants and clinical outcomes in inammatory bowel disease. Expert Rev Gastroenterol Hepatol 9:497–505

Hirscheld GM, Dyson JK, Alexander GJM etal (2018) The British

Society of Gastroenterology/UK-PBC primary biliary cholangitis treatment and management guidelines. Gut 67(9):65–74

Husby S, Koletzko S, Korponay-Szabo IR etal (2014) European Society

for Paediatric Gastroenterology, Hepatology and Nutrition guidelines for the diagnosis of celiac disease. J Pediatr Gastroenterol Nutr 54:136–160

John M.Eisenberg Center for Clinical Decisions and Communications

Science (2016) Diagnosis of celiac disease: current state of the evidence. Baylor College of Medicine, Houston, TX, Issued: July 26,

2016. Agency for Healthcare Research and Quality AHRQ USA.

https://www.ncbi.nlm.nih.gov/books/NBK379842/pdf/Bookshelf_ NBK379842.pdf. Accessed 12 December 2016

Kavanaugh A, Tomar R, Reveille J etal (2000) Guidelines for clinical

use of the antinuclear antibody test and tests for specic autoantibodies to nuclear antigens. Arch Pathol Lab Med 124:71–81

Kupfer SS, Jabri B (2012) Celiac disease pathophysiology. Gastronintest

Endosc Clin N Am 22(4):639

Leonard MM, Camhi S, Huedo-Medina TB et al (2015) Celiac dis-

ease genomic, environmental, microbiome, and metabolomics (CDGEMM) study design: approach to the future of personalized prevention of celiac disease. Nutrients 7:9325–9336

Lindor KD, Gershwin ME, Poupon R etal (2009) Primary biliary cir-

rhosis. Hepatology 50:291–308

Melegari A, Bonaguri C, Russo A etal (2012) A comparative study on

the reliability of an automated system for the evaluation of cellbased indirect immunouorescence. Autoimmun Rev 11:713–716

Mitrev N, Leong RW (2017) Therapeutic drug monitoring of anti

tumor-necrosis factor α agents in inammatory bowel diseases. Expert Opin Drug Saf 16(3):303–317

Nakamura M, Komori A, Ito M et al (2007) Predictive role of anti-

gp210 and anticentromere antibodies in long-term outcome in primary biliary cirrhosis. Hepatol Res 37:5412–5419

Nielsen OH, Ainsworth MA (2013) Tumor necrosis factor inhibitors for

inammatory bowel disease. N Engl J Med 369:754–762

Ordás I, Mould DR, Feagan BG et al (2012) Anti-TNF monoclonal

antibodies in inammatory bowel disease: pharmacokinetics-based

dosing paradigms. Clin Pharmacol Ther 91:634–626 Plebani M, Tozzoli R, Trenti T etal (2017) Appropriatezza della richi-

esta di esami ed esiti clinici: il caso delle malattie renali, tiroidee e

della celiachia. Biochim Clin 41:266–285 Porcelli B, Alessio MG, Villalta D etal (2015) Linee guida per la diag-

nosi di laboratorio e istologica della malattia celiaca. Revisione

2015. Riv Ital Med Lab 11:76–95

Porcelli B, Cinquanta L, Barberio G etal (2016) Quanto e come ven-

gono applicate le linee guida in Autoimmunologia nei Laboratori

italiani? Riv Ital Med Lab 12:221–233 Rigby WF (2007) Drug insight: different mechanisms of action of

tumor necrosis factor antagonists passive-aggressive behavior? Nat

Clin Pract Rheumatol 3:227–233 Rossi E, Basso D, Zambon CF etal (2015) TNFA haplotype genetic

testing improves HLA in estimating the risk of celiac disease in

children. PLoS One 10:e0123244. https://doi.org/10.1371/journal.

pone.0123244

Schaeverbeke T, Truchetet ME, Kostine M etal (2016) Immunogenicity

of biologic agent in rheumatoid arthritis: lessons from clinical prac-

tice. Rheumatology 55:210–220 Strassburg LP (2010) Autoimmune hepatitis. Best Pract Res Clin

Gastroenterol 5:667–682 Thalayasingam N, Isaacs JD (2011) Anti-TNF therapy. Best Pract Res

Clin Rheumatol 25:549–567 Tighe D, McNamara D (2017) Clinical impact of immunomonitoring in

the treatment of inammatory bowel disease. World J Gastroenterol

23:414–425 Tonutti E, Bizzaro N (2014) Diagnosis and classication of celiac dis-

ease and gluten sensitivity. Autoimmun Rev 13:472–476 Tozzoli R, Bizzaro N, Tonutti E etal (2002) Guidelines for the labora-

tory use of autoantibody tests in the diagnosis and monitoring of

autoimmune rheumatic diseases. Am J Pathol 117:316–324 Van der Windt DA, Jellema P, Mulder CJ etal (2010) Diagnostic test-

ing for celiac disease among patients with abdominal symptoms: a

systematic review. JAMA 303:1738–1746 Vande Casteele N, Gils A, Singh S etal (2013) Antibody response to

iniximab and its impact on pharmacokinetics can be transient. Am

J Gastroenterol 108:962–971 Villalta D, Sorrentino C, Girolami E etal (2015) Autoantibody proling

of patients with primary biliary cirrhosis using a multiplex line-blot

assay. Clin Chim Acta 438:135–138 Vincent FB, Morand EF, Murphy K et al (2013) Antidrug antibod-

ies (ADAb) to tumour necrosis factor (TNF)-specic neutralising

agents in chronicinammatory diseases: a real issue, a clinical per-

spective. Ann Rheum Dis 72:165–178 Wiik AS (2003) Appropriateness of autoantibodies testing in clinical

medicine. Clin Chim Acta 333(2):177–180

Laboratory Diagnostics inAllergic

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AldaTizianaScacchetti andTommasoTrenti

Introduction

Currently, a considerable amount of evidence seems to indicate that the increased susceptibility of allergic individuals to produce allergen-specic IgE is under the control of multiple genetic and environmental factors. Although IgE is the immunoglobulin class quantitatively less represented in humans, the interaction between these antibodies, allergens, and effector cells gives rise to an impressive cellular immune reaction, which can trigger the well-dened clinical picture of allergic diseases.

In the past decades, immunoallergological diseases have registered a constant increase in their prevalence, sometimes with abrupt accelerations; they are characterized by heterogeneous and evolving clinical pictures.

Based on this general description, allergies could be considered an epidemic due to their inexorable progression, especially in the most industrialized areas of the world. Indeed, they are not contagious, yet they seem unstoppable; in most cases, they do not seriously jeopardize survival but weigh on the quality of life; they are treatable but sometimes not curable in their dynamic chronicity.

Discovery ofImmunoglobulin E

The discovery of immunoglobulin E (IgE) in the late 1960s allowed the use of specic biomarkers of allergy in the diagnostic process. IgE was discovered in 1967 during the studies of Gunnar Johansson and Hans Bennich in IgE myeloma. It is structurally made up of two heavy chains and two light chains.

A. T. Scacchetti Department of Laboratory Medicine and Pathological Anatomy, Baggiovara Hospital, Modena, Italy

T. Trenti ( Department of Laboratory Medicine and Pathological Anatomy, Azienda USL/Azienda Ospedaliero-Universitaria di Modena, Modena, Italy e-mail: t.trenti@ausl.mo.it

The possibility of having large quantities of puried IgE, obtained from the few cases of IgE myelomas, has allowed us to obtain, through animal immunization, puried anti-IgE antibodies, with consequent development of radioimmunological and enzyme immunoassay methods, which are now widely used in the diagnosis of allergic diseases.

Subsequent research has essentially focused on studying the molecular and cellular basis of the IgE response and subsequent IgE-mediated reactions, identifying the various chemical mediators, preformed or neoformed, and on the denition of their pathogenetic role in immediate and late reactions and the subsequent induced allergic inammation.

Mast Cells andBasophils

IgE-binding cells were identied by incubating human leukocytes and tissue cells with iodine-125 (125I)-labeled myeloma IgE or with 125I-labeled IgE-specic antiserum. In both cases, autoradiography made it possible to demonstrate that the labeled probes used in the studies conjugated to blood basophils and tissue mast cells.

Basophilic granulocytes, in humans, represent 0.5–1% of circulating leukocytes. Their granule-rich cytoplasm is stained by basic dyes, hence the name of basophils. Under the electron microscope, they are characterized by a plurilobed nucleus, relatively few mitochondria, numerous glycogen granules, and membrane-bound electron granules distributed throughout the cytoplasm.

Paul Elrich rst described mast cells in 1877. They were named so in reference to the numerous granules mistakenly believed to be exogenous and ingested by the cell. Mast cell precursors are formed in the bone marrow during hematopoiesis and then migrate to all peripheral vascularized tissues, where they differentiate into mature cells. They are present in the connective tissue contiguous to blood and lymph vessels and, in high numbers, in the skin and mucosal surfaces of the respiratory and gastrointestinal tracts.

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Allergens

First, the terms “allergenic source,” “allergenic extract,” and “allergen” should be appropriately dened.

The term allergen source refers to the material container of allergens; for example, peach, dog, egg, and milk are allergen sources.

Allergenic extracts, commonly used in invivo and invitro laboratory diagnostics, come from dened allergenic sources (peach, dog, mites, grass pollen, etc.) and are obtained by extraction and purication processes. Subsequently, the extracts are standardized according to different methods in the various diagnostic industries, with consequent results that are not comparable with each other due to the present lack of a harmonization process.

The quality of these extracts has improved substantially over the years; however, it still presents criticalities and limitations that are difcult to eliminate. The criticalities are intrinsically linked to the extraction processes, which cause the loss of some allergenic proteins, the acquisition of proteins from unknown sources, and the different concentrations and protein composition from one batch to another. Only recently, the concentrations (expressed in μg/mL) of major allergenic proteins have been measured in the extracts, but not of minor proteins, which might even be absent. The absence or low concentration of allergenic proteins in the extract may cause false negatives during diagnosis and consequent ineffectiveness of hyposensitizing therapies when the proteins contained in the extract are not present at the concentrations necessary to induce desensitization.

The results of in vitro specic IgE according to the extracts used vary according to the type of extract used, and the method used (CAP, Thermo Fisher Scientic; Immulite, Siemens etc.) and are therefore not comparable. To these variables, which can be dened as “dependent on the diagnostic technology,” must be added those related to the clinical symptoms, the age of the patient, the time when the tests are performed (onset of the disease or follow-up), the prevalence of allergy in the population studied, the clinical complexity of the allergic pathology, and its globality.

The limitation, so far insuperable, consists in the impossibility of establishing, in a patient showing polysensitization to specic IgE invitro, whether the polysensitization is due to cosensitization (sensitization to distinct and unique molecules from different allergen sources) or a mechanism of co-recognition (sensitization to different allergen sources containing homologous molecules).

The term allergen refers to a protein, glycoprotein, or carrier-conjugated haptene, with a molecular weight of 5–150kDa and an isoelectric point between 2 and 10, capable of binding specic IgE and inducing an allergic reaction.

Thus, each allergen source contains different allergenic proteins, and each allergen may have a different number of antigenic determinants or epitopes.

An epitope is dened as an amino acid sequence recognized by a specic antibody; for example, milk-specic IgE recognizes specic epitopes contained in milk.

Generally, epitopes can be distinguished into linear, when IgE recognizes a contiguous amino acid sequence in the primary structure of the antigen, and conformational, when IgE recognizes a noncontiguous amino acid sequence characteristic of the three-dimensional structure of the protein (Fig.37.1).

The primary sequence of an allergen can be easily found in online search sites such as Allergome (AllergomeAligner,

www.allergome.org/ script/tools.php?tool = blaster) or

BLAST in UniProt (www.uniprot.org).

The structural folds of a protein are of primary importance in provoking immunological sensitization and the relative antibody response. Many allergenic proteins, if subjected to heat or the action of proteolytic enzymes, as occurs during food preparation or the digestive process, undergo modications that can determine the loss of conformational epitopes but also the possible unmasking of linear epitopes.

Food allergens can be divided into:

• Class 1 food allergens: these are made up of proteins

resistant to digestion and heat and can act as sensitizers in

the gastrointestinal tract. To this class belong, for exam-

ple, the major allergenic proteins of milk, egg, sh, crus-

taceans, and some vegetables.

epitope

Linear

epitope

Conformational

Fig. 37.1 Conformational epitopes consist of noncontiguous amino acids in the protein’s primary structure, but they to be adjacent in the tertiary structure due to the folding of the protein chain. Their formation, therefore, depends on the three-dimensional structure of the allergen. Linear epitopes, on the other hand, are made up of amino acids that are adjacent in the primary structure of the protein. (Copyright EDISES

2021. Reproduced with permission)

Linear

epitope

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• Class 2 food allergens consist of proteins that are not resistant to heat and digestion and are generally incapable of causing systemic symptoms. They are present in plants and foods of animal origin (thermolabile proteins of milk, meat, and eggs) and cause symptoms mainly localized to the oral cavity (oral allergy syndrome) as they lose their antigenic power following degradation in the stomach. The symptoms appear after sensitization to homologous allergens contained in pollens (nonsensitizing elicitors). This phenomenon, dened as cross-reactivity, explains why some patients can present even severe reactions when taking allergenic foods never before ingested.

The allergenicity of a single protein, therefore, depends on:

• its epitopes;

• its spatial conformation upon exposure to antigenprocessing cells, such as macrophages, dendritic cells, or B lymphocytes;

• avidity (degree of reaction) between IgE and epitopes, which in turn depends on the number of allergenic epitopes on the molecule (valence), the size, and conformation of the molecule;

• degree of afnity between antibodies and epitopes, which increases in the course of the humoral immune response.

• The fourth letter (in lower case, as dened by the nomenclature of living organisms) indicates the rst letter of the second name of the allergenic source. For this reason, a molecular component of Phleum pratense is dened as Phl p;

• A number is added to the letters to distinguish each component from all the others: Phl p1 indicates the rst component identied (and usually cloned) in Phleum pratense;

• Other numbers can be used to dene the component further: for example, Amb a 1, from Ambrosia artemisiifolia, has some isoallergens: Amb a 1.01, Amb a 1.02, Amb a

1.03, and Amb a 1.04. For Amb a 1.01, three different variants have been described (Amb a 1.0101, Amb a

1.0102, and Amb a 1.0103), characterized by a very high homology in the primary sequence;

• Finally, the letter “r” or “n” preceding the name of the component indicates its origin (r for recombinant or n for natural). Recombinant components are allergens cloned into eukaryotic or prokaryotic vectors using genetic engineering techniques; when a component is produced in prokaryotes (e.g., Escherichia coli), it does not have glycosylated chains.

Extractive natural molecules are highly puried (in this

case, posttranslational modications, such as glycosylations, are present).

In recent years, 2503 molecular allergens have been characterized at the molecular level, and the latest update is January 6, 2017.

Identifying and characterizing allergenic sources have led to the subsequent industrial production and marketing of natural allergens puried or produced with recombinant DNA technologies.

The recombinant molecules thus obtained have a sensitivity of over 70% in mimicking the allergenic source.

Allergenic molecules are divided into genuine, true markers of a specic source (e.g., Ole e 1 is the marker protein of allergy to olive tree pollen and other Oleaceae), and panallergens, proteins shared by allergenic sources even taxonomically unrelated to each other, responsible for apparent polysensitization to tests performed with extracts. For example, prolin is a panallergen shared by pollens and plant foods. Its recognition by a patient allergic to pollens will cause positivity to all types of pollens and plant foods tested without the patient experiencing symptoms upon exposure to them.

The naming of the components observes an international convention:

• The rst three letters (such as Phl, Bet, etc.) correspond to

the rst three letters of the Linnean name of the allergenic

source (in the example, Phleum, Betula);

Use ofRecombinant Allergens inAllergology Diagnostics

The use of recombinant (or highly puried native) allergens to replace allergenic extracts represents a remarkable achievement in allergology as it improves the clinicaldiagnostic process for several reasons. The rst is that it allows overcoming one of the most critical hurdles related to the use of allergenic extracts, which is that of standardization, and this is because of their variability in composition and antigenic content, the diversity of supply sources, the presence of proteolytic enzymes and contaminated allergens, etc. Recently, the WHO/IUIS standardization committee (www.allergen.org) has embarked on a new program to introduce new standards from puried or recombinant native proteins, to be distributed to industries or academic bodies to prepare invitro tests or to regulatory bodies for the comparison of allergen products. Using these standards allows for dening the allergenic content in mass units, which, if it can have a relative meaning in laboratory diagnostics, has an essential meaning in the preparations for specic immunotherapy. Moreover, the standard obtained using recombinant molecules has the advantage of being able to be reproduced unmodied and in practically unlimited quantities over time. Another essential advantage of molecular diagnostics is the

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ability to discriminate whether a state of polysensitization identied invivo or invitro, using extractive preparations, is due to cosensitizations, i.e., primary sensitizations toward specic major or minor allergenic proteins present in single allergenic sources, or to cross-sensitizations, i.e., crossreactivities toward homologous molecules present in different allergenic sources, sometimes without clinical signicance. The most important advantage, however, is at the level of diagnostic denition. A diagnosis based on traditional allergenic extracts can only lead to the identication of the allergenic source (e.g., allergy to birch, mites, etc.), but not of the molecular entity to which a patient is sensitized (e.g., Bet v1, Bet v2, Bet v4, Der p1, Der p10, etc.). Identifying the individual allergenic prole of a patient is not only a diagnostic renement, but it has signicant repercussions both prognostic and therapeutic, as described later.

In Vitro Allergy Diagnostics

The use of invitro tests by various methods introduced in recent decades has undoubtedly led to signicant progress in diagnosing IgE-mediated allergic syndromes.

Among the advantages of these tests, globally considered, we should point out above all the remarkably high sensitivity and specicity, the good reproducibility, the absolute harmlessness for the patient, and the possibility of being researched even while taking antihistamine therapy; the disadvantages are summarized, in practice, in the relatively high cost.

Detection ofTotal IgE

The determination of total IgE was introduced many years ago with the PRIST (Paper RadioImmunoSorbent Test) and is currently performed by various automated methods.

The serum concentration of total IgE varies, in non-atopic adults, from 10 to 200IU/mL; in infants, this concentration is only a few IU/mL, and it progressively increases and reaches adult levels around the tenth year of life.

Total IgE is generally high in allergic syndromes due to IgE-mediated immunoreactions. Notably, the highest values of IgE are found in allergic diseases, but the nding of “normal” values of total serum IgE does not exclude the diagnosis of allergic disease. Indeed, many allergic patients have total IgE levels within the normal range.

It should also be mentioned that total IgE is found with altered values in various pathological conditions that are certainly not allergic and physiological or paraphysiological conditions (e.g., in smokers).

Therefore, it can be concluded that the determination of total IgE alone is of little clinical signicance in allergic diseases.

Detection ofSpecic IgE

Since 1967, the year in which the rst specic IgE assay was performed by Wide and collaborators, with the birth of the RAST (RadioAllergoSorbent Test), much progress has been made in the eld of laboratory allergology diagnostics, through various stages consisting of the development of technologies based on solid phases with high binding capacity; use of enzymatic tracers linked to monoclonal IgE antibodies with calibration curves made with standards of known titre in quantitative units kUa/L and produced according to an international reference standard (now 3° IS WHO 11/234, formerly 2° IRP WHO 75/502 for total IgE); the introduction of diagnostic systems for the determination of specic IgE with high analytical sensitivity in total automation (third generation test).

Over the years, RAST has been followed by numerous other methods, which have replaced radioactive markers, and the techniques developed have been numerous (ELISA [Enzyme Linked ImmunoSorbent Assay], agglutination, precipitation). These invitro methods differed signicantly in terms of the type of detection system (colorimetric, uorimetric, and chemiluminescence), the antiserum (mono or polyclonal, single or mixed), the support for carrying the allergen (cellulose polymers, polystyrene spheres, etc.), and the development in a solid or liquid phase, resulting in signicant inhomogeneity among the different systems available in the laboratory setting. Since not all systems are the same, and not all are equally valid, the laboratory must guarantee the reproducibility of the data (system with a coefcient of variation <15%– NCCLS, 2004) and is certied by a national and/or international quality control (QC) program. Currently, the most used methods for detecting specic IgE are Cap FEIA and 3gAllergy.

Cap FEIA onImmunocap

It uses an immunouorenzyme method and is based on a solid phase (cellulose polymer) covalently conjugated with the allergen, which reacts with the serum under examination so that the specic IgE, if present, binds to the allergen. After washing to remove unbound IgE, i.e., not specic for the allergen, labeled anti-IgE antibodies are added. Thus, a “sandwich” complex consists of the solid phase with allergen + patient-specic IgE+labeled anti-IgE.Using the uorimetric detection method, the amount of marker present in the complex is measured, which will be directly proportional to the amount of specic IgE present in the sample under examination.

3gAllergy onImmulite 2000

It uses an enzyme-enhanced chemiluminescent method and is based on the use of a liquid phase. Patient serumcontaining specic IgE and liquid allergen are incubated in

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the presence of antiligand coated beads. The liquid allergen, which acts as a ligand, binds to the specic IgE in the serum and the antiligand- coated beads. The ligand-specic IgE is detected by an anti-IgE antibody conjugated to an enzyme that catalyzes the chemiluminescent reaction in the presence of its specic substrate. Also, in this method, the concentrations obtained are proportional to the amount of specic IgE present in the patient’s serum under examination.

Molecular Allergy Diagnostics

Starting from the mid-nineties of the last century, in parallel with the development of proteomic techniques, we have witnessed the birth of molecular allergology, which has provided various conrmations and numerous new ndings in the eld of allergology. In particular, the following must be mentioned:

• Most allergens possess high antigenic complexity.

• The individual response to an allergen depends on the genetic background of each subject. Several allergens can present structural homology, which can determine phenomena of cross-reactivity due to the recognition of different allergens by the same antibody.

• IgE generally tends to recognize conformational and nonlinear epitopes. Indeed, in the case of food allergies, only molecules that maintain their three-dimensional structure even at high temperatures can induce allergy in sensitized patients.

The availability of recombinant allergens and/or highly

puried extracts has allowed the introduction of molecular diagnostics (Component Resolved Diagnosis, CRD), which allows the analysis of IgE reactivity to individual allergens.

CRD allows the characterization of the specic allergo-

logical prole of an individual, allowing the distinction between primary or genuine sensitizations and crossreactivities. The CRD has, therefore, revolutionized the clinical management of the patient allowing, for example, the selection of patients who can benet from specic immunotherapy (ITS), or the assessment of the severity of the reaction to food allergens, allowing to distinguish between subjects sensitive to highly stable molecules and subjects sensitive to thermal and gastro-labile molecules, which will develop only oral allergy syndrome (OAS) after ingestion of raw food.

Analytical Methods forMolecular Diagnostics

CRD can be performed through the use of:

• Single molecular components (singleplex), which allow a targeted diagnosis

• Matrices, which consist of many allergens deposited on microarrays (multiplex)

Singleplex Molecular Diagnostics

Monoplex molecular diagnostics consists of a second-level test to conrm a diagnostic suspicion, allowing the identication of specic IgE toward a specic allergen. It has the advantage of being performed on the same analytical platform (quantitative, highly automated) where specic IgE toward extractive allergens are sought; this allows the use of algorithms by a reex test approach so that the execution of few and targeted analytical assays determines an appropriate and efcient use of the available economic resources. In this approach, however, there is a risk of underestimating the presence of other unsuspected sensitizations by identifying only the components hypothesized a priori. The principle of the method is the same as the immunoassays described above for Cap FEIA and 3gAllergy consolidated on the same instruments used for the research of specic IgE, except allergens that in the determination of allergenic molecules will be recombinant or native allergens.

Multiplex Molecular Diagnostics (Microarrays)

Microarray diagnostics is a third-level test that allows the denition of the allergy prole of a patient. Currently, a microarray (ImmunoCAP ISAC, Thermo Fisher Scientic, Waltham, MA, USA) is available on the market that allows the simultaneous determination of IgE directed toward more than a hundred different allergens using a small amount of serum (30μL). However, interpreting this test is somewhat complex and requires highly specialized personnel.

The test is also semi-quantitative, calibrated against an

internal standard, and has lower diagnostic accuracy than the Cap FEIA test.

Table 37.1 describes the main characteristics of the two

diagnostic systems. From the point of view of purely productive efciency for the resources used, diagnostics using the microarray, based on the current costs envisaged for carrying out the tests, is advantageous if the patient’s clinical situation requires the search for specic IgE toward several molecular components greater than 12–13 allergens.

Microarray Diagnostics (ImmunoCAP® ISAC). Fluorescent antihuman IgE antibodies are used to detect

antigen–antibody binding between specic IgE present in the patient’s serum and antigens conjugated to a solid phase on a slide (chip). The uorescence is subsequently measured by a scanner equipped with a laser excitation source. A densitometry software then analyzes the image and provides the test results as a function of the uorescence intensity detected on each spot.

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Table 37.1 Advantages and disadvantages of ISAC multiplexes and Cap FEIA singleplex

Advantages Disadvantages

ISAC

30μL of serum or plasma from capillary or venous blood 112 allergens that can be measured simultaneously Natural and recombinant proteins Less amount of allergen required No interference due to high total IgE concentrations

Cap

Automated method

FEIA

Quantitative results High sensitivity Low coefcient of variation Natural or recombinant proteins or crude extracts Appropriate for monitoring sensitization

Manual method Semi-quantitative results Less sensitivity For certain allergens, a high interassay variability has been reported Increased coefcient of variation Some allergenic sources are not included Inappropriate for monitoring sensitization Potential interference between IgE and other isotypes, mainly IgG

Each allergen is individually dosed Detection of low afnity antibodies which may have little clinical relevance

The test takes approximately 5h to perform. The results are processed as ISAC classes (absent-low-medium-high) and international system units, providing a semi-quantitative IgE determination based on a specic reference curve. The system has high diagnostic reliability as each molecule is tested in triplicate.

Microarray-based diagnostics could be particularly useful in the diagnostic framing of complex clinical situations, such as:

• Patients with multiple sensitizations, not identiable

based on history and rst-level tests

• Patients who do not respond to ITS therapy for whom it is

appropriate to evaluate the presence of other possible

allergens

• Patients with “idiopathic” anaphylaxis, to identify any

sensitization not diagnosed by traditional tests

• Pediatric patients for whom it is not possible to obtain a

sufcient sample to perform the analysis by monoplex

diagnostics

However, there are some limitations to the use of multiplex technology:

1. The test is not automated and, therefore, is rather com-

plex to perform and subject to a higher risk of errors.

2. Reduced analytical sensitivity and specicity character-

ize it compared to the single-plex method and a high rate of false positives.

3. The interpretation of the results is complex and must be performed by a specialist with a good knowledge of the various molecules and their diagnostic signicance.

A new test system (ALEX2, MacroArrayDX, Wien, Austria) has recently been introduced for simultaneous detection of Total IgE and Specic IgE to 117 extracts and 183 molecules by solid-phase enzyme immunoassay. Extract and allergen molecules combined with nano-particles are sorbed on a solid-phase substrate, forming a macroscopic multiplex matrix - the immune allergy chip.

Conclusions

Molecular diagnostics in allergology has been a real revolution, having modied and transformed the concept of allergy, introducing a new language with a different semiological approach to diagnosis and therapy. The reference to allergenic families and their molecules, rather than to allergenic sources (plants, trees, fruit, etc.), can create quite a few difculties in interpreting the diagnostic information produced, in particular, and understandably, for general practitioners, but sometimes also for clinical specialists in the eld. For these reasons, the professionals working in the allergology laboratory must have the necessary skills to transfer the diagnostic information aimed at the efcacy of the results obtained from molecular tests in a clear language to the requesting physician and, possibly, to the patient himself. The interpretative comment of the invitro molecular tests is a fundamental moment in the diagnostic process of the allergic patient.

The allergy diagnostic laboratory is fundamental in guaranteeing an effective diagnostic process of allergic pathologies only if it can receive adequate clinical information associated with the request for specic IgE. Without this information, the use of allergenic extracts and allergological molecules risks entering the great range of inappropriateness, generating not so much and not only an increase in costs but also wrong diagnoses or, even worse, delayed and confusing diagnoses.

The presence of an anamnestic card with the patient’s clinical information substantially improves the research and selection of allergens to be prepared in the diagnostic session. The clinical pathologist must evaluate each request for specic IgE in light of the prescriber’s request. This evaluation leads to a patient-specic choice of which panels or individual allergens to test. Based on the test result obtained with allergen extracts, the clinical pathologist with allergological expertise may decide to integrate the test with molecular allergens if this increases the informative value of the report. At the end of the diagnostic procedure, the clinical pathologist can produce an interpretative report that

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illustrates the results obtained and the diagnostic pathway used. This practice is undoubtedly burdensome in terms of time required, but it is certainly the most effective to optimize costs and resources and ensure patients’ health and the overall quality of the clinical diagnostic-therapeutic process.

The evolution of allergy diagnostics has now arrived at a nal and personalized mapping of the allergic patient. Although the molecular frontier is complex, it will become or already is a tool of fundamental importance in the armamentarium of the clinical allergist. An educational and training process of continuous updating with the interaction between laboratory medicine professionals and clinical allergology specialists is of paramount importance for the effective and appropriate use and interpretation of the diagnostic information produced by these innovative technologies and analytical methods.