General Aspects in Diagnosing

Autoimmune Diseases

Angie Lyn Acot, RMT, MPH ©

2018
Objectives:
Day 1
 Pre test
 Define autoimmune disease
 Identify two groups of autoimmune disease
 Laboratory diagnostic used to identify AI
 IFA Protocol (Running)
 ANA
 Hep-2 pattern classification
 ANA test recommendation
 QC Used (Internal and External)
 Post test
 Day 2 (Whole Day)

 Hand on training in Running IFA

 Hands on reading of IFA Patterns under the microscope
 Exam in identification of ANA patterns
Autoimmune disease
• Disease in which the pathology is caused by immune responses to
self antigens of normal cells and organs causing them to be
damaged.
Autoimmune diseases

Detection!!
• There are two different groups of Autoimmune
diseases:
– Organ-specific autoimmune diseases: are
characterized by immune-mediated injury localized
to a single organ or tissue. We find diseases as type 1
diabetes in which the organ injured is pancreas or
thyroid in case of Hashimoto Thyroiditis.
– Non-organ-specific autoimmune diseases
(Systemic AI Diseases, Connective Tissue Diseases
(CTD)): characterized by immune reactions against
many different organs or tissues, resulting on
widespread injury as in Systemic Lupus
Erythematosus (SLE)
Autoimmune diseases

 There are more than 80 autoimmune diseases with low

prevalence and incidence:
• Systemic diseases (CTD):
– Systemic Lupus Erythematosus
– Rheumatoid Arthritis
– Vasculitis
– Antiphospholipid Syndrome
• Organ-specific autoimmune diseases (OSAD):
– Diabetes Type I
– Celiac disease
– Thyroiditis (Grave´s disease)

 Often, patients with CTD develop other CTDs -> Development of

other autoantibodies (For example RA & Sjögren)
 Often patients with OSAD develop other OSAD (For example
Celiac disease & Diabetes Type I)
 Not clear causes:

 Infectious diseases: by bystander T-cell activation or molecular

mimicry (Coxsackie virus, CMV…)
 Vaccines (adjuvants)
 Genetics
 Hormones (sex)
 Hygiene ¿¿??
 Complex diagnosis:
 Most of the affected subjects initially have modest, nonspecific
clinical signs
 Overlap of clinical symptoms.
 Laboratory tests can help to make the correct diagnosis
DIAGNOSIS OF AUTOIMMUNE DISEASES

• Clinical exam & symptoms:

– Clinical history
– Manifestations

• Laboratory findings:
– Detection of autoantibodies
– Others (complement, Lupus anticoagulant, inflammation
markers, immunoglobulin levels…)

• Other tests:
– Radiology
– Histopathology
– Immunopathology
Diagnostic Techniques
 There are two principal techniques for the detection of autoantibodies:
• Immunofluorescence (IF):
 Gold Standard for ANA testing
 Detects more than 150 possible autoantibodies in one single test.
• ELISA:
 Important in diagnosis of organ specific autoimmune diseases.
 Confirmation in ANA – Only detects limited amount of autoantibodies

 The ELISA is inside the group of SOLID PHASE assays

 Diagnostic Techniques

 Other Solid Phase Technologies:

 Immunoblot
 Chemiluminiscence
 Multiplex (bead based)
 Diagnostic Techniques
 Some auto-antibodies are accepted as a marker of an
autoimmune disease.
 Some of them could be used also as early markers of prognosis
 There are several techniques to detect their presence in patient
serum, but all of them are based basically in having the antigen
responsible of the immune reaction immobilized in a surface.
The kind of surface where the antigen is immobilized is the
difference between the different types of techniques.
Antigen

Surface
Diagnostic Techniques

 In all the techniques the serum is incubated in contact with the antigen:
 If the serum has antibodies (positive serum) against this antigens they will
bind them and will remain immobilized in the surface after washing.
 If the serum do not contain these antibodies (negative serum) they will not
bind them and will be removed in washing step.

Human auto-
antibody (in
serum)

Antigen

Surface
Diagnostic Techniques

 After sample incubation and washing a secondary antibody (anti-human-

antibody antibody) labeled with a molecule that can be detected (fluorophore,
enzyme, etc...) (conjugate) is incubated in the surface.
 If the sample is positive there will be human antibodies bound into the surface and the
conjugate will bind this antibodies.
 If the sample is negative the conjugate will be removed during the washing.
Conjugate
(secondary antibody)

Human auto-
antibody (in
serum)

Antigen

Surface
Diagnostic Techniques

 IFA (IMMUNOFLUORESCENCE ASSAY): The antigen is native, without

purification, present in cells or tissue adhered in the slide. The
conjugate is labeled with fluorescein isothiocyanate (FITC), a
fluorescent molecule.

• Native antigen
FITC
(whole cell or
tissue)
• Detection of
more than one
autoantibody ?
type per test
Diagnostic Techniques - ELISA

 ELISA (ENZYME LINKED IMMUNOSORBENT ASSAY): A purified antigen

is coated into a well where the reaction is done. The conjugate is
generally labeled with HRP (Horseradish Peroxidase) that produces
yellow color in presence of TMB (3,3',5,5'-tetramethylbenzidine).

• Purified Antigen TMB

HR HR
coated to the P P
ELISA plate
P
• Detection of
only one
autoantibody ?
type per test
Diagnostic Techniques

Fibrillarin
positive serum

Negative result

Positive result

SSA Fibrillari M2
n
SSA Scl 70

ELISA IFA
Autoimmune diseases
ELISA
 High Throughput
 Automatic result reading
 Automatable process
Objective result
 Only detects a limited number of
autoantibodies
 Detects antibodies of high and low
avidity
 The antigens purification can damage
epitopes and protein folds
 Result variability between brands
 
COMMODITY/HIGH THROUGHPUT
IFA
 Screening of more than 150
autoantibodies in one single test
 Cheap
 It preserves the epitopes and protein
structures
 It only detects high avidity antibodies
 It takes some time to read results: not
automatable
 Subjective result
 High specialized personal needed
 Result variability between brands
RELIABILITY
IFA Protocol
1. PREPARE PBS: Dilute
1 bottle of PBS
(100mL) in 1L of
distilled water.
IFA protocol
2. IDENTIFY THE
DILUTION TUBES
CORRECTLY:
Dillution and Sample
ID

3. DILUTE THE
SAMPLES IN PBS:
The controls are
ready for use
 IFA PROTOCOL
4. OPEN AND IDENTIFY
THE SLIDES

5. DISPENSE THE
CONTROLS AND
SAMPLE DILUTIONS
IN THE SLIDE: Try not
to touch the well with
the pippette. 25uL for
cells and 50uL for
tissues.
 IFA PROTOCOL

6. PUT THE SLIDES IN

THE MOISTURE
CHAMBER

7. INCUBATE FOR 30
MINUTES
 IFA PROTOCOL
8. REMOVE THE
EXCESS OF SAMPLE
WITH PBS

If the slide has 2 lines,

first remove one line
and then the other.
Avoid mixture of
different samples
between wells
 IFA PROTOCOL

9. WASH THE SLIDE

WITH FRESH PBS
FOR 5 MINUTES
TWICE

10. DRY THE SLIDE

WITH THE BLOTTING
PAPER: Avoid
touching the wells
 IFA PROTOCOL
11. APPLY THE
CONJUGATE:
Dispense enough drops
to cover the well.

12. PUT THE SLIDES IN

THE MOISTURE
CHAMBER
 IFA PROTOCOL

13. INCUBATE FOR

30 MINUTES

14. REMOVE THE

EXCESS OF
CONJUGATE WITH
PBS
 IFA PROTOCOL

15. WASH THE SLIDE

WITH FRESH PBS
FOR 5 MINUTES
TWICE

16. APPLY THE

MOUNTING
MEDIUM: It´s
important to avoid a
lack or an excess of
mounting medium
 IFA PROTOCOL
17. PUT THE COVER
SLIDE. Try avoid
bubbles.

18. READ THE RESULT IN

THE MICROSCOPE
 IFA PROTOCOL
HEp2 CELLS nDNA

TISSUES
Examples: ANCA

• ASMA (Anti-Smooth
Muscle Antibody)

• LKM (Liver-Kidney-
Microsomal)
AUTOIMMUNE DISEASES DIAGNOSIS
Systemic AI diseases

Angie Lyn Acot, RMT, MPH ©

2018
ABBREVIATIONS
Disease Abrev.
SYSTEMIC AUTOIMMUNE DISEASES
Connective Tissue Diseases CTD
Systemic Lupus Erithematosus SLE
Drug Induced Lupus DIL
Rheumatoid Arthritis RA
Sjögen Syndrome SjS
Dermatomyositis DM
Polimyositis PM
Sistemic Sclerosis / Sclerodermia Scl / SS
Limited Sclerodermia CREST
Antiphospholipid Syndrome APS
Mixed Connective Tissue Disease MCTD
ORGAN SPECIFIC DISEASES
Autoimmune Hepatitis AIH
Primary Biliary Chirrosis PBC
Celiac Disease CD
Inflammatory Bowel Disease IBD
ABBREVIATIONS
PRODUCT ABREV.
Anti-Nuclear Antibodies ANA
Anti- Neutrophil Cytoplasmic Antibodies ANCA
Anti-Endomysium Antibodies AEA
Autoantibodies RL/RK/RS Triple
Anti-Mithocondrial Antibodies AMA
Anti-Smooth Muscle Antibodies ASMA
Glomerular Basement Antibodies GBMA
Anti - Adrenal Antibodies AACA
Anti-Striated Muscle Antibodies AStMA
Anti-Islet Cells Antibodies AICA
Anti-Thyroid Antibodies ATA
Anti-Skin Antibodies ASA
Anti-Keratin Antibodies AKA

PATTERN ABREV.
Liver - Kidney Microsomal LKM
Anti-liver-cytosol type 1 LC1
Anti-Mithocondrial Antibodies AMA
Anti-Smooth Muscle Antibodies ASMA
Anti-Nuclear Antibodies ANA
Anti-Reticulin Antibodies ARA
 CTD Diagnosis
 The most popular test for CTD is Antinuclear Antibodies
(ANA).
 Other IFA test such as nDNA, ANCA are also used.
 Some solid phase (for ex. ELISA) specific tests can also be used
for detection of specific diseaseS.
ANTINUCLEAR ANTIBODIES
(ANA)
ANA
• The most usual procedure for CTD diagnosis is:
• ANA Screening Over HEp2 (Gold Standard)
• Titration of positive samples (semi-quantitative)
• If positive, ANA confirmation in solid phase
immunoassay (usually ELISA). If possible, antigen
specificity is determined.
 ANA
 The gold standard for ANA detection is Immunofluorescence
over HEp2 since it can detect more than 150 possible
autoantibodies (2009 - ACR Recommendations on ANA testing)
ANA
 Care should be taken when interpreting positive results.
Autoantibodies could be found in:
 Elderly people: The 10-15% of healthy individuals of more than 65
years old has ANA positive at = or < than 1/160
 Pregnant women
 Patients with tumors
 Chronic infections
 Unspecific inflammations
 Others
 The presence of ANA in a patient without symptoms have a low
predictive value (only 11% of predictive value)
 A positive result needs to be used in conjunction with other
evidence and clinical judgment
ANA testing

 Over 35 patterns are described from more than 150 different

known antigens.
 Patient sera frequently contain different autoantibodies that
result in mixed patterns.
 It´s normal to develop new autoantibodies during an
autoimmune disease (more than one disease can be developed).
 ANA testing
 Nuclear homogeneous, nuclear coarse speckled, and nuclear
centromeric patterns appear exclusively in patients with AI
diseases
 Other patterns can appear in other conditions:
 Cancer
 Infections
 Inflammations
 Etc…
 So… A positive result does not mean an autoimmune disease and
a negative result does not rule out an autoimmune disease!
HEp2 CELLS

ANA – Why HEp-2?

ANTI-NUCLEAR ANTIBODIES (ANA HEp-2): HEp-2 Cells
(derived from laryngeal carcinoma cells )

HEp-2 cells have a huge

nucleus (about 80% of total cell
volume) thus being specially
useful for Antinuclear Antibodies
testing
HEp-2 cells show a high mitotic
rate, allowing an easier
identification of specific
structures
HEp-2 cells growth in
monolayer until maximum of
confluence is achieved
ANA – HEp2
• In ANA-HEp2 the test for autoantibodies is negative if no
specific pattern is recognized
• If patient specimen is positive then determine the titer by
serially diluting the sample.
• The titer is the highest dilution demonstrating a 1+ specific
fluorescence of the substrate.
ANA – HEp2
• The screening dilutions should be defined locally
• In general:
– 1/40 for children under 16 years old
– 1/80 or 1/160 for adults
– 1/160 for people > 60 years old

Dillution % “False” Positive results

1:40 20 – 30%
1:80 10 – 20%
1:160 5%
1:320 3%
 ANA – HEp2

 Many laboratories reports the IFA ANA-HEp2 cells results as 5

or 6 basic patterns:
 Homogeneous
 Speckled
 Nucleolar
 Centromere
 Cytoplasmic
 Negative
 Other laboratories decide to include other patterns such as
NuMa, nuclear dots, membrane etc…
 There are more than 30 patterns described
HEp2 Pattern Interpretation
Pattern of resting cell

Pattern of mitotic cell

+ MIT
- MIT

Set HEp2 pattern Homogeneous Speckled

CELL CYCLE
HEp2 Pattern Interpretation
HEp2 Pattern Interpretation
 NEGATIVE The methaphasic plate is negative or the same
MITOSIS fluorescent intensity when compared with the
HEp2 Pattern Interpretation
(MIT -) surroundings

To set if the mitosis is positive or negative we always compare the

fluorescence intensity of the chromosomes and the “cytoplasm”

More intense fluorescence located in the

POSITIVE
methaphasic plate (or where the chromosomes
MITOSIS
are supposed to be) when compared with the
(MIT +)
surroundings
BASIC PATTERNS
Basic patterns

 Homogenous Nuclear pattern

 Speckled Nuclear pattern
 Centromere pattern
 Nucleolar pattern
 Cytoplasmic pattern
 Negative
HEp2 PATTERNS CLASSIFICATION
Homogeneous nuclear pattern
HEp2 PATTERNS CLASSIFICATION
2.2.2 Fine speckled nuclear pattern
HEp2 PATTERNS CLASSIFICATION
2.2.1 Large speckled nuclear pattern (nuclear matrix)
 HEp2 PATTERNS CLASSIFICATION
2.2.2 Coarse speckled nuclear pattern
2.2.6 Centromere pattern
2.2.7 Multiple nuclear dots
2.2.7 Few Nuclear Dots
3.1 Homogeneous nucleolar pattern
3.2 Clumpy nucleolar pattern
3.3 Punctuate nucleolar pattern
1.1 Smooth membranous nuclear pattern
1.2 Punctuate membranous nuclear pattern
2.2.5 Pleomorphic speckled nuclear pattern (PCNA)
5.6 Actin-like pattern
5.7 Vimentin-like pattern
5.4 Lysosomal-like pattern
5.1 Fine speckled cytoplasmic pattern
5.3 Mithocondrial-like cytoplasmic pattern
5.5 Golgi-like pattern
4.1 Centriole spindle pattern (centrosome)
4.3 Spindle fibre pattern
4.2 Spindle pole pattern (NuMa)
4.4 Midbody pattern (MSA)
 ANA test recommendations
 The ANA test should be ordered only when the patient´s history,
symptoms and physical examination findings are suggestive of a
connective tissue disease.
 The laboratory profile can be individual test or search of a
general suspected disease
 It´s important for better interpretation of the result to have
patient data (age, sex, history and other laboratory tests results)
 ANA test recommendations
 Ideally use serum that have been collected within a few hours
 Storage at 4ºC up to a week after collection
 Storage at -20ºC for longer periods (2 weeks – 1 thawed only)
 Frequent thawing and freezing of the sample may lead to a
decrease of autoantibody activity
 ANA test recommendations
 Internal and External quality controls should be used:
• Internal quality control: use known positive (pattern and
titre) and negative serums. In IFA put positive in first well and
negative in second well to assure not crossed contamination.
• External quality control: blind samples given by external
company. Assures the reliability of results in the laboratory.
THANKS FOR YOUR ATTENTION!