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Laboratory Diagnostics inAutoimmune
https://t.me/medicina_free
Diseases
TommasoTrenti, AlessandraMelegari,
andChiaraBonaguri
36
Introduction
The detection of circulating autoantibodies is crucial for
autoimmune diseases, from diagnosis to prognosis and
treatment.
Autoimmune phenomenology, one of the most studied
topics in the eld of immunology, has been the subject of
many clinical, laboratory and experimental studies in the last
decades, which have led to important clarications about the
immune physiopathology and have allowed to group in the
same pathogenetic class many diseases whose cause was
previously uncertain.
Nowadays, there are an increasing number of diseases for
which a pathogenesis linked to abnormalities of the Immune
Competent System has been ascertained or only hypothesized and which are often characterized by the presence of
immunoglobulins in the circulation of autoantibodies,
directed against “self” substances represented by constituents of the organism itself.
Autoimmune diseases develop when the immune system
fails to recognize the “self” and activates cells or produces
antibodies directed against cells, tissues and/or organs of the
organism, causing an inammatory process with tissue damage and consequent expression of an autoimmune disease.
The pathogenetic role of immunological reactions resulting
from this loss of competence in the recognition of the “self”
together heterogeneous pathological conditions in terms of
clinical features represents the common denominator of diseases with autoimmune etiology.
T. Trenti (*)
Department of Laboratory Medicine and Pathology, Azienda USL/
Azienda Ospedaliero-Universitaria di Modena, Modena, Italy
e-mail: t.trenti@ausl.mo.it
A. Melegari
Department of Laboratory Medicine and Pathology, Azienda USL
of Modena, Modena, Italy
C. Bonaguri
Laboratory of Clinical Chemistry and Hematology, University
Hospital of Parma, Parma, Italy
The natural history of all autoimmune diseases is divided
into different phases or conditions, which can be basically
ascribed to a potential phase, a latent subclinical phase and a
clinical phase.
Until now, more than 80 pathologies have been described
that derive from an autoimmune response with an estimated
prevalence in Europe of 4000 affected persons per 100,000
inhabitants and an estimated 2,400,000 patients in Italy.
The availability of serological diagnostic tests, consisting
of a panel of increasingly specic and sensitive biomarkers,
has allowed the laboratory to play a role, not only of reference as central but irreplaceable in the diagnosis and prognosis of these diseases, resulting the laboratory medicine the
only discipline that consider the autoimmunity in its globality, while, in the various clinical areas, appears divided by
organ or specialty.
Indeed, the skills of the laboratory professional in relation
to the entire clinical-diagnostic pathway are essential to offer
an effective response to the request of the clinician, aimed at
ensuring the health needs of the patient, and these skills must
be performed with autonomy in the choice of diagnostic
investigations useful to identify the evolution of autoimmune
diseases in its complex and multiple expressions.
The remarkable progress in immunological diagnostics
that has occurred in recent years, linked to the increased
physiopathological knowledge of the role of autoantibodies
and the development of new analytical technologies, together
with the growing expectations of physicians and patients for
investigations, has created the need for a more appropriate
use of immunological tests for the detection of autoantibodies, both in terms of request and execution of the same.
Appropriateness is a complex objective that can only be
achieved by integrating skills and agreeing on the best procedures to follow in each specic clinical situation. If, nally,
appropriateness of requests and services also means less
waste and better use of available resources, it is evident once
again that this goal can be achieved more effectively, not so
much through budgeting operations or restrictive economic
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. Ciaccio (ed.), Clinical and Laboratory Medicine Textbook, https://doi.org/10.1007/978-3-031-24958-7_36
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measures but through collaboration between laboratory physician and clinician.
This chapter will discuss some autoimmune diseases and
the related laboratory diagnostic procedures established
according to the most recent recommendations and clinical
evidence, which are continuously updated.
In this chapter, we present autoimmune rheumatic diseases, monitoring of biological drugs, celiac disease and
autoimmune liver diseases, which have been selected in
order to outline through the individual presentations the
diagnostic complexity and the new promising perspectives
that in the eld of laboratory medicine affect the discipline of
autoimmunity.
It was decided to present these themes because they are
very emblematic of how much discussion, comparative
activity and government there has been and still is and how
the laboratory is the reecting mirror that has gone through
many changes and rened in recent years all its ability to
diagnose in close collaboration with scientic research and
technological instrumentation.
In fact, in autoimmunology, besides the indirect immunouorescence technique, which keeps an irreplaceable role,
new immunoassay tests, different in analytical technique
(enzyme immunoassays, chemiluminescence immunoassays, immunoblots, etc.) but all potentially endowed with
opportunities for results standardization, are now routinely
used for single or multiple autoantibody assays and/or are
currently object of numerous studies.
In this scientic context, which will probably see in the
coming years the emergence of new diagnostic strategies and
solutions, with consequent therapeutic effects and needs for
clinical governance of processes, the role in terms of expertise of the autoimmunologist laboratory will be essential.
Autoimmune Rheumatic Diseases
The detection of pathology-specic antibody proles is a key
diagnostic criterion for the characterization of patients with
connective tissue disease.
The most used serological tests are the detection of antinuclear antibodies (ANA), extractable nuclear antigens
(ENA), and anti-dsDNA. The current nomenclature and
acronyms for ANA and ENA are being re-evaluated; however, due to their widespread use, they will be retained as
such in this chapter.
In recent years, the demand for these tests has increased
signicantly, due in part to the acquisition of new evidence
on their clinical relevance but also to an inappropriate prescription, not well quantied, for:
• Insufcient communication between clinicians and labo-
ratories, resulting in disagreement on the diagnostic sig-
nicance of available tests and test results
• Different analytical techniques available for the same
marker, availability of new target antigens
• Lack of both homogeneous terminologies and diagnostic
algorithms, resulting in inhomogeneous behaviour
The in-depth evaluation of the clinical signicance and
diagnostic rationale of the laboratory’s ANA, ENA and
dsDNA tests is at the heart of the approach to connective tissue disease, identifying screening tests and in-depth tests
that may allow an appropriate request to the clinical
question.
The presence of ANA in serum is considered a marker of
high diagnostic signicance in collagen diseases.
The positive ANA test represents, in fact, a classicatory
criterion in the diagnostic denition of non-organ-specic
autoimmune diseases, such as systemic lupus erythematosus
(SLE), scleroderma/progressive systemic sclerosis (SSc),
mixed connective tissue disease (MCTD), Sjogren’s syndrome (SS), dermatomyositis-polymyositis (DM/PM) and
undetermined connective tissue disease (UCTD).
On the contrary, rheumatoid arthritis (RA) does not
express this marker: This pathology frequently presents a
serum negativity towards the autoantigens commonly evaluated in the diagnosis of connective tissue diseases. For this
diagnosis, the combined evaluation of rheumatoid factors
(RATest) and anti-cyclic citrullinated peptide antibodies
(anti-CCP) is of particular interest.
Therefore, the detection of ANA in the serum of patients
is an important diagnostic tool to allow the clinician a more
precise classication of anamnestic and clinical pictures that
are often blurred.
In recent years, commercial test offerings in this eld
have become numerous as the analytical techniques available
for the same marker.
It is, therefore, necessary to make a reasoned choice of
diagnostic tests and analytical methods in which the laboratory must be able to play a central role.
ANA are autoantibodies belonging to all classes of immu-
noglobulins, most often to the IgG class, directed against cellular constituents common to all cell types (non-organ-specic
antibodies).
The study of the structural and functional characteristics
of the target antigens and the characterization of the single
antigen/antibody systems has made it possible to dene close
correlations between serological picture and clinical status,
and in some conditions, ANA represent a real marker of disease, which, due to the precocity of appearance, assumes a
high predictive and prognostic value.
Due to its high diagnostic sensitivity, the ANA test exe-
cuted IFI method remains the gold standard and represents
the rst level of the diagnostic algorithm.
However, since these autoantibodies, especially at low
titre, are also found in healthy subjects and/or those with
non-autoimmune diseases, it is necessary to perform the
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ANA test and the related in-depth examinations (ENA and
dsDNA) according to a correct sequence to improve the
diagnostic process.
Based on all the above considerations, the application of
the ANA Reex is justied. The ANA Reex allows the
autoimmunology laboratory to evaluate the uoroscopic pattern, the positivity titre and the clinical information and to
assess whether and which tests should be performed as indepth examinations.
In order to be effective, the ANA Reex must be an
administrative tool valid for all prescribers both in hospitals
and in the territory, but with the freedom to be applied in a
“reexive” way by the laboratory, which becomes responsible for the governance of the whole subsequent process.
In the presence of a positive ANA-IFI result, a greater
diagnostic and prognostic power can be achieved with the
characterization of antibody specicities directed against the
different nuclear antigens through molecular tests.
The determination of specic antibodies directed towards
the so-called ENA-, DNA- or RNA-associated proteins is
useful for the diagnosis of SLE, neonatal lupus, SS, MCTD,
PM/DM and SSc, with variable sensitivity and specicity, as
reported in the literature. The variety of autoantigens recognized by ANA is extremely wide.
Anti-ENA antibodies in serum can be detected by several
techniques currently used in the laboratory: enzyme immunoassay (ELISA), immunoblot (IB), and immunoassay (EIA).
For the choice of the method, it is necessary to keep in
mind some factors: clinical location (reference laboratory or
not), laboratory budget and, consequently, cost of reagents,
organization and “experience” of the laboratory and level of
dialogue between laboratory and clinician.
The ideal method should meet the criteria of clinical sensitivity, precision and accuracy, ease of execution, easy availability and low cost; at present, there is no method that can
meet all these requirements alone.
Considering the characteristics of the available methods,
a correct diagnostic procedure for the detection of anti-ENA
can only make use of several methodological approaches
that the autoimmunology laboratory must be able to put in
place.
The diagnostic and prognostic usefulness of the antidsDNA antibody assay has led to the development of several
techniques for their identication: radiobinding techniques
(Farr technique), indirect immunouorescence (IFI) on
Crithidia Luciliae and enzyme immunoassay techniques
(ELISA).
Anti-dsDNA antibodies are highly specic for SLE as
they are almost exclusively found in SLE and are the tenth
criterion for SLE according to the American College of
Rheumatology.
If their concentration is high, they are prognostic of a
relapse even if SLE is quiescent; moreover, their presence,
in the absence of clinical symptoms, is indicative of subclinical SLE, being negative in drug-induced SLE and positive in less than 2% of cases with other autoimmune
diseases. Numerous studies have documented that the concentration of anti-dsDNA antibodies correlates with the
clinical course of SLE and lupus nephritis and that an
increase in antibody titre may precede clinical are by a
few weeks.
The research of ANA in IFI is penalized by several critical
issues, such as the subjectivity of the reader, the qualication
and experience of the reader, the variability related to different substrates and the technology of the microscope. All this,
together with the consolidation of the laboratories, has led to
a workload of microscopic activity that is not always sustainable where there is a shortage of personnel dedicated to autoimmune diagnostics and there is no generational turnover
and no insertion and training of new professionals. These
problematic aspects have seen, in recent years, the emergence of new organizational and technological strategies to
cope with the increasing numbers of tests and the contraction
of healthcare personnel.
Recently, automated reading systems have been introduced in the laboratories, which mainly allow a screening
between negative and positive samples and among the latter
a “potential” attribution of the patterns of positivity and an
estimation of the titre with the possibility of archiving the
images as a partial solution to the problems mentioned
above. The technological offer is of platforms with different
characteristics that, however, must be dropped in their own
organization, taking advantage of the benets of standardization that, however, come only after an insertion and an evaluation that requires an alignment with their own microscopic
practice. Certainly, the possibility of archiving images and,
more generally, the integration of these systems in the management island of autoimmunity can make the daily work
more efcient.
The scientic literature has produced several comparisons
and considerations on the systems on the market, analyzing
their strengths and weaknesses. Also these technologies, like
the others, must be consciously included and governed by a
structured and robust experience in the eld of
autoimmunology.
Considering the objective difculties in numbers and
organization, studies of comparative evaluation of ANA in
IFI and solid phase ANA in immunoassay have also
increased. There are different technologies and combinations
of antigens on the market. It is a question of considering the
comparison and possible combination of two different
screening approaches. There are studies on the combination
of the two tests in the clinical diagnostic pathway, and there
are studies on the use of the solid phase as initial screening,
followed by immunouorescence. In addition to sensitivity
and specicity reasoning, it is necessary for each laboratory
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to evaluate its own population, informatics and organizational strategy and clinical algorithm. Certainly, these
choices, if shared across a regional territory, increase the
strength of an algorithm.
Monitoring ofBiological Drugs
Therapeutic Monitoring ofAutoantibodies
andTNFα Inhibitor Drugs inPatients Being
Treated forChronic Immune-Mediated
Inammatory Diseases
In the last decades, the therapeutic approach to chronic
immune-mediated inammatory diseases has been revolutionized by the advent of biological drugs, monoclonal antibodies capable of neutralizing the pro-inammatory and
immune-activating effect of tumour necrosis factor alpha
(TNFα), the main cytokine involved in the pathogenesis of
these diseases. This class of drugs includes chimeric (iniximab), humanized (adalimumab, golimumab), fusion protein
(etanercept) and recombinant humanized antibodies (certolizumab). They are monoclonal antibodies with different
structural and functional characteristics, different pharmacokinetic and pharmacodynamic properties and different efcacy and therapeutic safety proles.
It is estimated that about 60–70% of patients treated with
these drugs achieve favourable therapeutic responses in the
phase of induction and maintenance of clinical remission of
the disease, while the remainder experience therapeutic failure due to inefcacy of the therapy, loss of response over
time or the appearance of adverse events, such as infections
and reactions at the site of infusion. The main cause of therapeutic failure is the phenomenon of immunogenicity.
Different epitopes of the anti-TNFα molecule are recognized
as foreign elements by the immune system, which initiates
the production of antibodies directed against the drug itself
(antidrug antibodies, ADA).
ADAs belong to different classes of immunoglobulins
(IgG, IgE, IgM) and are able to form a complex that prevents the binding of the drug to TNFα with a consequent
increase in clearance and reduction in the functional concentration of the drug, which leads to a loss of response to
therapy. ADAs can, therefore, interfere both at the pharmacokinetic level, reducing the active concentrations of the
anti-TNFα drug, and at the pharmacodynamic level, preventing the drug from binding to TNFα to neutralize it.
Biological drugs have different degrees of immunogenicity
that vary depending on the route of administration, molecular structure, dose and any concomitant therapies. In general, the increase in ADA formation is associated with a
reduction in the concentration of anti-TNFα drug measurable in the bloodstream.
In order to facilitate the most appropriate therapeutic
choices and try to prevent immunogenicity, loss of response
to therapy and the occurrence of adverse events, in recent
years have been developed different schemes of therapeutic
drug monitoring (TDM) specic for each disease, including
the determination of the concentration of biological drug in
use and the search for ADA.
From the point of view of the laboratory, the TDM presents some criticalities concerning: the methods of determination of the TDM concentration of the drug and ADAs,
measurement of the concentration of the drug in the bloodstream and assessment of the presence of ADAs.
To date, the most widely used methods in the laboratory
for the determination of TNFα inhibitory drugs and for the
detection of ADAs are ELISA and the RIA method, but
there are also other liquid phase enzymatic tests and tests
that measure mobility, such as the Homogeneus Shift Assay
(HSMA). There is no difference between liquid or solid
phase assays in terms of measured concentration of the
drug, but a difference has been observed in terms of specicity. The main limitation of these methods is the risk of
having false positive results, due to the non-specic binding between the immunoglobulin and the drug. In the
absence of standardization between the different tests,
which contributes to limiting the reproducibility of the
results, it is advisable to always monitor the patient with
the same analytical method.
With regard to the determination of the concentration of
the drug in circulation, the aspects that should always be
taken into consideration are: (1) the sampling time, that is the
number of days between the last infusion and the drug determination; (2) the number of previous infusions; (3) the characteristics of the patient that may inuence the circulating
levels of the drug (weight may modify the clearance of the
drug, as well as gender, BMI value or concomitant use of
immunosuppressants); (4) the characteristics of each drug
(e.g. the kinetics and the route of administration, intravenous
or subcutaneous). Moreover, drugs have specic trough levels and different concentration ranges depending on the type
and duration of the disease and on the individual characteristics of the patients. For therapeutic monitoring purposes, it is
useful to dene therapeutic windows within which drug concentrations should fall.
The search for and determination of ADAs represent a
critical aspect of TDM, especially since the factors associated with their development are not yet fully understood.
ADAs can be neutralizing and non-neutralizing depending
on the action they mediate on the drug itself. In fact, they
can inhibit the anti-inammatory action of the drug by
binding directly to it, or they can form immune complexes,
reducing the bioavailability of the drug by increasing its
clearence. A distinction is then made between bound and
free ADAs. The former are bound to the drug, while the
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free ones in circulation are detected when all the binding
sites of the drug are saturated and, consequently, the concentration of free ADAs that could be determined is
reduced.
Two other aspects must also be kept in mind in TDM:
• ADAs can be transient. In patients for whom minimal
amounts of ADA are detected, these may disappear in a
short time and, therefore, do not affect the activity of
TNFα inhibitors; conversely, in the presence of high concentrations of ADA, these are usually suggestive of an
ongoing immunogenic process;
• ADAs may give rise to cross-reactions with biosimilar
drugs and may be interfered with by rheumatoid factor.
Finally, the lack of a recognized and validated cut-off
makes the interpretation of the laboratory data difcult.
Although the TDM of biologic drugs for autoimmune dis-
eases presents some criticalities to date, the determination of
the concentration of the drug in the bloodstream and ADAs
is important at all stages of therapy, from induction and
maintenance phase to remission, in order to ensure that
patients receive adequate therapy and reduce the risk of loss
of response.
Celiac Disease
The celiac condition is an autoimmune enteropathy (the most
frequent autoimmune pathology since about 1% of the population presumably suffers from it) caused by the ingestion of
cereals containing gluten and represents the nal phase of a
series of complex processes that involve both innate and
adaptive immunity. It is believed that the prevalence of this
disease is underestimated, since for every patient diagnosed,
3–10 subjects affected by the disease are not recognized,
even though the diagnosis of the disease is signicantly
increasing both in Europe and in the United States, doubling
every 15years or so. The possibility of developing the disease is due to the presence of cofactors that cause the disease
to appear at a particular time in the lives of predisposed individuals. Therefore, contrary to what was previously thought,
this is not a prevalently paediatric disease; on the contrary,
most subjects develop it in adulthood, and in 20% of the
cases, the onset and diagnosis occur after the age of 60. From
a clinical point of view, the manifestations of the disease are
extremely variable and include pictures ranging from severe
deterioration of general conditions (rare) to nuanced clinical
symptoms (frequent), often with the absence of classic gastrointestinal symptoms. In children, the symptoms are usually more specic: gastroenterological symptoms (pain,
abdominal bloating, etc.), irritability and mood swings and
developmental decits, etc., whereas in adults, the disease
often presents in a nuanced manner or is detected
incidentally.
From a pathogenetic point of view, what happens when
gliadin penetrates the intestinal barrier is sufciently well
known. The damage occurs in the proximal tenue with complex alterations that occur at the moment of gluten introduction, affecting both innate immunity (with the production of
IL-15) and adaptive immunity (with the activation of the
immune system). The result is an alteration of biohumoral
and cellular factors that cause both functional and anatomical deterioration of the intestinal villi with pictures up to
complete atrophy. If the celiac condition is the only autoimmune disease for which an environmental trigger, gluten (a
prolamin contained in wheat where, however, there are other
toxic prolamins, present in spelt, kamut, rye and barley), is
recognized, genetic predisposition is of extraordinary importance. Environment and genes, however, are necessary but
not sufcient elements for the appearance of the coeliac condition: 30% of the general population has predisposing genes
and comes into contact with gluten, but only 1% develops the
celiac condition. There are groups at increased risk of developing the disease: rst and second degree relatives of celiac
persons, individuals with selective IgA deciency, individuals with other autoimmune diseases or genetic abnormalities.
For rst-degree relatives, the risk is 10–15 times higher than
for the general population. The conclusive diagnosis of the
coelic disease may be proposed on the basis of clinical ndings and the positive results of two intestinal biopsies, but
now greater importance is being attributed to serological
markers and genetic tests as they are of adequate diagnostic
accuracy, cost efcacy and are widely used. Research and
scientic evidence have questioned the value of the intestinal
biopsy as the “gold standard,” while the diagnostic, serological and genotyping tests are increasingly robust, although a
joint evaluation of clinical, serological, genetic and histological data is necessary when present and deemed
necessary.
Genetics intheDiagnosis ofCeliac Disease:
Role ofHLA DQ2 andDQ8
The genetic background is a necessary condition for the
development of the disease in both the classic clinical forms
and the latent and silent forms. The main genetic factor predisposing to celiac disease is the HLA DQ2 haplotype
(DQA1*0501-DQB1*0201), which is expressed in 90% of
patients affected by the celiac condition, while the DQ8 haplotype (DQA1*0301-DQB1*0302) is expressed in 5% of
patients and the remaining 5% are carriers of at least one of
the two DQ2 allies (usually DQB1*0201). In a very concise
manner, it can be stated that approximately 70–75% of the
population is not a carrier of DQ2 or DQ8 and, therefore, has
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very little, if any, chance of suffering from the celiac condition. Of the remaining 25–30% of the population who are
carriers of DQ2 or DQ8, only 4% will develop the disease.
Therefore, the presence of the DQ2 or DQ8 haplotype identied by a positive genetic test is a necessary, but not sufcient, condition for diagnosing the celiac condition.
HLA DQ2/DQ8 molecules are expressed in the dendritic
cells of the gut mucosa and have high afnity for deamidated
gliadin peptides; the binding between DQ2/DQ8 and deamidated peptides is the basis of the immune response as glutenderived antigens are exposed to T cells that trigger the
pathological process of autoimmune response; this is the reason for the importance of the HLA DQ2/DQ8 genetic
conguration.
HLA class II includes not only DQ2 and DQ8 but a wide
variety of possible alleles, many of which are involved in
susceptibility to autoimmune diseases or, in some cases, are
protective. HLA enters the major histocompatibility complex (MHC) locus on the short arm of chromosome 6, where
several genetic regions are located; the one of interest in
celiac disease is the class II region, which contains a number
of genes, including HLA DQB1 and HLA DQA1, which are
in linkage with the DR locus. The A1 gene encodes for the α
protein chain, the B1 for the β
presenting cells, these proteins are expressed at the membrane level to form a heterodimer. About DQ2, the most
important allele is *02 of the DQB1 locus, which encodes
the β chain of the HLA DQ2 heterodimer. For the presence
of DQ2 for the expression of celiac disease, it is necessary to
have this allele. However, it is not sufcient: it is also necessary to have the *05 allele of the DQA1 locus. These alleles
can be combined in a very variable and complex way. For
DQ8, the combination of the *0302 allele from the DQB1
locus and the *03 allele from the DQA1 locus is required.
Many techniques, now routine in the molecular biology laboratory, are available to perform HLA DQ2/DQ8 analysis. For
example, an allele-specic PCR can be performed with analysis by agarose gel electrophoresis, or sequencing can be
performed. In reality, however, the goal is to detect only the
possible presence of the four alleles directly involved. One of
the possible methods is a real-time PCR analysis, using specic sequences, primers and probes that recognize the alleles
of interest. The DNA sample is analyzed, and if the main
allele (*02 of the DQB1 locus) is detected, an amplication
signal is detected. In order to conrm that the subject is positive for HLA DQ2, it is then necessary to detect also the *05
allele of the DQA1 locus, using the same method. It is then
possible to verify if *02 of the DQB1 locus is in homozygosity or in heterozygosity. If the result is positive, the various
allelic combinations translate into a different risk of disease.
In fact, it is a different condition to have all the DQ2 and
DQ8 alleles expressed compared to having only *02 of the
DQB1 locus.
protein chain; in antigen-
As far as family members are concerned, who are individuals at risk, there is debate in the scientic community
about the advisability of carrying out the genetic test: If it is
negative, the celiac condition can be excluded, and this is a
reason for supporting the advisability of the test. However,
the probability that family members of celiacs have this haplotype and that the test is positive is very high, and in this
case, there is nothing that can be done to prevent the disease.
The problem, therefore, is to translate the different risk associated with the results of the genetic analysis into clinical
practice. There are many other genes other than the HLA
genes mentioned above that play a role in the predisposition
to celiac disease and other autoimmune diseases, but each
non-HLA variant makes a minimal contribution to the
genetic risk associated with HLA; therefore, it is difcult to
think of analyzing all the variants, and, in any case, their
clinical signicance is not particularly relevant.
In conclusion, with regard to the diagnosis of celiac disease due to genetic predisposition, it can be concluded as:
• The HLA locus is the main genetic predisposition factor
for celiac disease.
• The presence of the HLA DQ2 and DQ8 haplotypes is
necessary, but not sufcient for the development of the
pathology.
• The absence of HLA DQ2 and HLA DQ8 allows the
exclusion of celiac disease, and these patients do not
require further testing over time for suspected celiac
disease.
• There is a risk gradient for celiac disease associated with
the different HLA DQ haplotypes that are derived from
various combinations of the HLA DQA1 and DQB1
alleles.
• The conclusive diagnosis of celiac disease in symptom-
atic children is made if there is the simultaneous presence
of HLA DQ2 or DQ8 with IgA class antitransglutaminase
levels greater than 10 times the threshold used by the
laboratory.
Biomarkers andAlgorithms forDiagnosis
andMonitoring ofCeliac Disease
It is essential to emphasize that all the diagnostic tests
required for the diagnosis of the celiac condition must be
carried out on an unrestricted diet: It is not necessary to suspend the intake of gluten before undergoing the diagnostic
procedure in order to distort the results of the tests. For the
patient, once the diagnosis has been made, an aglutinated
diet followed regularly is fundamental; in fact, the elimination of gluten from the diet makes it possible to re-establish
an intestine that is structurally almost normal and functionally
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intact. The tolerated amount of gluten is 10mg/day. Early
diagnosis of the pathology in a celiac subject, even if he or
she is almost asymptomatic, is very important, since important complications may appear over time. The most signicant of these are refractory celiac disease (a condition in
which malabsorption symptoms and villi atrophy persist
even when the strict aglutination diet has been in place for at
least 6–12 months), ulcerative jejunalitis and collagenous
sprue (which involve high mortality), lymphomas and epithelial tumours. The risk of developing a complication of the
latter type has been reduced over time, thanks to the possibility of identifying (and then subjecting to the gluten-free diet)
even subjects with milder forms than in the past, characterized by very nuanced symptoms.
The biomarkers of rst choice for the diagnosis of celiac
disease are IgA class anti-transglutaminase antibodies (antitTG IgA) and total IgA, where total IgA is performed mandatorily in the case of the rst intake of the patient to assess
any decit of the same for potential false negatives. In the
case of IgA deciency, IgG class deamidated peptide antibodies (anti-DGP IgG) or IgG transglutaminase antibodies
are considered. While determination of plasma concentration
of anti-tTG IgA antibodies is generally considered more
appropriate, detection of anti-DGP-IgA antibodies remains
useful in children under 2 years of age or in cases of IgA
deciency.
Important national and international guidelines as well
as national legislation now propose a denitive diagnosis of
the celiac condition using only biohumoral data without
performing an intestinal biopsy, in particular when values
greater than 10 times the threshold value of the method’s
specic anti-tTG IgA antibodies are determined. In this
case, the positive predictive value of the test is equal to that
of biopsy in paediatric patients when associated with positivity for anti-endomysium EMA antibodies and HLA
DQ2/DQ8 genetic testing. The European Society of
Paediatric Gastroenterology, Hepatology and Nutrition
(ESPGHAN) guidelines indicate that in symptomatic children with anti- tTG antibodies above tenfold threshold, conrmed by EMA and HLA DQ2–8 positivity, the diagnosis
is conclusive and biopsy can be avoided (Fig. 36.1).
Basically, the same diagnostic algorithm is applied in
adults, that is anti-tTG IgA antibodies and total IgA determination are the rst choice and initial tests, and anti-DGPIgG (anti-tTG-IgG) antibodies are normally performed
only in case of IgA deciency; the conrmation of the diagnosis in the case of positivity of the initial tests is obtained
by performing EMA antibodies and HLA DQ2/DQ8
genetic test. However, there is still the indication to perform intestinal biopsy in adult patients for a denitive conrmation of the diagnosis, or in any case, the primary
studies present are not conclusive to exclude the usefulness
of endoscopic examination. Genetic testing (presence of
HLA DQ2/DQ8 class II histocompatibility antigen) is recommended as a surveillance test for at-risk subjects, prior
to serology, because in the event of a negative result, the
problem of diagnosing the celiac condition will no longer
arise for the patient’s life; in the event of a positive result,
an investigation will be appropriate both in the event of
symptoms and in asymptomatic patients every 3 or 4years.
Anti-tTG
IgA + Total IgA
Positive anti-tTG IgA > 10 URL
+ Normal total IgA
Positive EMA
Positive HLA DQ2/DQ8
Celiac disease
diagnosis
Fig. 36.1 Algorithm for the diagnosis of celiac disease in subjects according to clinical manifestations and age. (Copyright EDISES 2021.
Reproduced with permission)
Negative EMA
Negative HLA DQ2/DQ8
Clinical and serological
monitoring
Endoscopy
Negative anti-tTG IgA
+ Normal total IgA
Positive anti-tTG IgA
< 10 URL + Normal
total IgA
Positive anti-tTG IgG
and anti-DGP IgG
investigations
Rule-out celiac
disease
Negative anti-tTG IgA
+ Deficiency total IgA
Required anti-tTG Ig
and anti-DGP IgG
Negative Anti-tTG IgG
and anti-DGP IgG
Stop laboratory
investigations
Rule-out celiac
disease
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For the follow-up of patients with celiac disease, the current ministerial guidelines recommend a check-up within
6–12months of diagnosis and, subsequently, every 1–2years
(except in the event of complications), with a blood count
and anti-tTG IgA (or IgG in the case of IgA deciency) at
each check-up. It is also appropriate to perform blood tests
for iron metabolism (sideremia, ferritin) and folatemia at the
rst check-up and repeat them at subsequent checks until
normalization in case of abnormal values. Since autoimmune
thyroiditis is frequently associated with celiac disease, TSH
and TPOAb should be determined at diagnosis, and if both
are negative, TSH should be repeated every 3years; if both
are positive, the patient should be taken to a specialist clinic
for autoimmune thyroid disease. Bone densitometry is recommended only in adults, after 18 months of gluten-free
diet. Other eventual haematological and instrumental tests
are recommended on the basis of the clinical evaluation.
Quality Characteristics ofAntitransglutaminase Antibodies Assessment
The tests for diagnosing the celiac condition still have very
differentiated and inhomogeneous analytical specications
where the process of harmonization has certainly not yet
been concluded. The negative predictive value of all these
diagnostic tests is rather high, but only anti-tTG class IgA
and anti-endomysium have such high specicity and sensitivity as to be considered satisfactory. Some comparative
studies have compared the EMA, anti-tTG and anti-DGP
serological tests: the test with the best characteristics in
terms of sensitivity and specicity appears to be the anti-tTG
IgA, followed by EMA.Regarding the determination of antitTG IgA, several diagnostic kits are commercially available,
which use different antigens, units and measurement ranges
and suggest different cut-offs. The most frequently used antigens are of recombinant human origin on E. coli, on
Baculovirus or derived from myeloma cell lines and puried
human-type antigens from erythrocytes. In children, studies
indicate that the diagnostic performance of anti-tTG IgA
assays is dependent on the method used and the age of the
child. The laboratory should indicate the antigen (recombinant or puried human), the method (enzyme immunoassay – ELISA, or chemiluminescence – CLIA) and the
diagnostic cut-off. In addition, it should dene and monitor
the analytical performance of the tests performed within its
scope. Regarding the antigen, a meta-analysis evaluated
diagnostic kits with different antigens, indicating that antigens of human origin, both recombinant and puried, have
superior specicity and sensitivity.
The heterogeneity of the results of the tests carried out in
the various laboratories was highlighted in 2009 by a study
whose results showed great variability in the sensitivity (69–
Table 36.1 Appropriateness of laboratory tests in diagnosising celiac
disease: Decalogue of recommendations, from Sibioc-Laboratory
Medicine
1. The diagnosis of celiac disease is based on the integration of
clinical, serological, and histological data. It is essential that all
diagnostic tests are carried out on a free diet, avoiding the
suspension of gluten intake, in order not to distort the results of
the tests.
2.
The biomarkers of rst choice in diagnosis are IgA antitransglutaminase antibodies (anti-tTG IgA) and total IgA.If
IgA deciency is detected, antibodies to gliadin deamidated
peptides (anti-DGP IgG) are measured.
3. The genetic test for the detection of HLA DQ2/DQ8 is indicated
for screening of subjects at risk before serology: Some experts
indicate its use at birth for rst degree relatives of celiac
subjects.
4. Diagnostic rst level tests for celiac disease in subjects with
clinical manifestations and over 2years of age are anti-tTG IgA
and total IgA.
5. The test of choice for subjects with IgA deciency are anti-tTG
IgG and/or anti-DGP IgG.
6. The most important test for asymptomatic individuals belonging
to risk groups is HLA DQ2/DQ8.
7. The EMA test in the diagnostic procedure remains
controversial.
8. In the diagnostic report of the biomarkers, the antigen used in
the method, the method and the diagnostic threshold value must
be clearly explained.
9. In symptomatic children with concomitant presence of HLA
DQ2/DQ8 and elevated levels of anti-tTG IgA (10 times the
URL), the diagnosis can be made without biopsy.
10. The absence of DQ2/DQ8in subjects at risk allows for
excluding celiac disease and to avoid its monitoring.
URL upper reference limit
93%) and specicity (96–100%) of the various methods and
a rather important coefcient of intra-assay variation, suggesting that a critical knowledge of the analytical methods in
use is necessary. With regard to the appropriateness of diagnostic tests in the diagnosis of celiac disease, a “decalogue of
recommendations” has been proposed by SIBioC-Laboratory
Medicine, see Table36.1.
Autoimmune Liver Diseases
The evolution of knowledge in the laboratory diagnostics of
autoimmune hepatopathies, and in particular of primary biliary cholangitis, well represents the profound changes that
have affected the eld of autoimmune disease diagnostics in
the last decade.
In recent years, in fact, molecular tests based on the use of
puried or recombinant antigens and new diagnostic strategies and solutions, some of which are automated, have been
added to the traditional methods in immunouorescence and
are currently the subject of different study protocols.
Autoimmune diseases of the liver are a heterogeneous
group of immune-mediated pathologies characterized by an
36 Laboratory Diagnostics inAutoimmune Diseases
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autoimmune aggression towards hepatocytes or
cholangiocytes and can be classied into three major entities, primary biliary cholangitis, autoimmune hepatitis and
primary sclerosing cholangitis, which contain within them
some variants as well as overlapping syndromes, so-called
Overlap, where both a cholestatic and an inammatory component is present to varying degrees. In this eld, the distinction between hepatocellular damage and cholestatic damage
is fundamental for the purposes of differential diagnosis, and
consequent therapeutic choices and antibody diagnostics
plays an essential role in the correct classication of the different nosological entities.
Autoimmune hepatitis (AIH) is a chronic inammation of
the liver of unknown cause, in which tolerance to the hepatocyte is lost. The mechanisms underlying liver damage are
cell-mediated and direct cytotoxicity.
The disease mainly affects females in both childhood and
adulthood and may be associated with other autoimmune
conditions (ulcerative colitis, autoimmune thyroiditis, primary biliary cholangitis and primary sclerosing
cholangitis).
It is usual to classify AIH on the basis of specic autoantibodies, which for type 1 are mainly ANA and anti-smooth
muscle antibodies (ASMA) (Fig.36.2); type 2, on the other
hand, usually presents LKM1 (liver-kidney microsomal antibodies). Other antibodies found in AIH are anti-SLA (soluble liver antigens), anti-LP (liver-pancreas), anti-ASGPR
(asialo-glycoprotein) and anti-LC1 (liver cytosol type 1).
Positivity for ANA, ASMA, LKM1 and SLA is included in
the diagnostic criteria for AIH.
Primary biliary cholangitis (CBP) is a relatively rare
chronic cholestatic hepatopathy with slow evolution and
autoimmune aetiology characterized by an aggression of T
lymphocytes against the cells of the intrahepatic bile ducts;
the consequent damage makes the drainage of bile from the
liver to the intestine difcult, thus causing stagnation in the
liver and resulting in liver failure in a variable time.
Patients with CBP are primarily female (90%), usually
diagnosed in the fth or sixth decade of life. It has been suggested that genetic susceptibility is a predisposing factor for
CBP, while environmental factors such as infections, chemicals and smoking may play a causal role.
The course of the disease is variable, and an accurate
diagnosis in the early stages is crucial as early drug treatment
can slow the progression to liver failure and improve
survival.
The diagnosis is based on a combination of clinical/histological features, abnormalities of the liver biochemical prole in a setting of persistent cholestasis for more than
6 months and the presence of AMA and/or specic ANA
antibodies in the serum.
AMA antibodies are detected in about 90% of CBP
patients, and their high sensitivity and specicity makes their
presence one of the three diagnostic criteria for CBP. The
AMA are actually a heterogeneous group of antibodies and
are directed with various percentages of frequency against
different autoantigens that are part of the structural complex
of the enzyme 2-oxyacid dehydrogenase, namely the subunits E2 of the enzyme pyruvate dehydrogenase (PDC-E2–
frequency 90%), the complex oxyglutaric dehydrogenase
Indirect Immunofluorescence Test
Fluoroscopic Pattern
ANA+ (homogenous)
Fig. 36.2 Fluoroscopic pictures relating to positivity for anti-nucleus (ANA +, left) and anti-smooth muscle (ASMA +, right) autoantibodies
Fluoroscopic pattern
ASMA+
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(OGDC-E2– frequency 50%) and branched-chain ketoacid
dehydrogenase (BCOAD-E2– frequency 50%).
AMAs can be detected in the serum of asymptomatic
patients with normal liver enzyme balance; follow-up studies
have shown that over time most of these subjects develop
CBP.
For a long time, the indirect immunouorescence test
(IIF) on cryostat sections of the liver, kidney and stomach of
rats was considered the gold standard for routine screening
of AMA.In recent years, on one hand, the limitations presented by this test (complex as execution, difcult to standardize, not fully automated, operator-dependent
interpretation) and, on the other hand, the identication of
molecular targets of AMA have led to the development, validation and introduction in the laboratory diagnostics of
molecular antigen-specic tests using the technique of
ELISA immunoassays or Immunoblotting.
The promising results obtained with the latest generation
of ELISA tests using the MIT3 antigen (PDC-E2, OGDC-E2
and BCOAD-E2), capable of detecting positivity in about
half of AMA IIF negative patients, suggest the use of these
tests in place of or alongside IIF for AMA testing, particularly when laboratories are unfamiliar with IIF use and
interpretation.
Immunoblotting assays also represent an interesting diagnostic alternative to the AMA in IIF because this multiplex
assay allows the simultaneous detection of different CBPspecic autoantibodies using recombinant antigens.
In addition to AMA, ANA, usually associated with autoimmune rheumatic diseases, are frequently found in CBP
patients. At present, the method considered the gold standard
for the determination of ANA is IIF, using HEp-2 cells as
substrate. CBP-specic ANA have been detected by several
authors in 30–50% of patients, and two different uorescence patterns have been described for these autoantibodies:
rime-like/membranous (specicity for gp210 and
Nucleoporin p62 proteins) and multiple nuclear dots (specicity for sp100, PML and small ubiquitin-like modier proteins). The detection of these antibodies, characterized by a
low sensitivity, allows to conrm the diagnosis of CBP in
AMA-negative patients, frequently liable to misclassication. There is also some evidence that the presence of specic CBP ANA, particularly anti-gp210, is associated with a
worse prognosis and a more aggressive disease. Finally, as
for AMA, also for specic CBP ANA, the new solid phase
tests (ELISA and Immunoblotting), especially those using
recombinant antigens, such as MIT3, appear to be more sensitive and less subjective than IIF (Fig.36.3).
Dot Blot method report
Session : New
Execution : 06/04/2012 12:02:33
Identifier
1. Pattern IFI nuclear dots
Antigen dot blot gp210+M2
Method : LIVER DOT BLOT 7 ANTIGENS
Lot
Indirect Immunofluorescence Assay (IFI)
2. Pattern IFI rhyme-like
Antigen dot blot gp210
User : primolab
3. Pattern IFI AMA+
Antigen dot blot M2
Fig. 36.3 Molecular antigens and analogous CBP-associated uoroscopic pictures
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