IMMUNOLOGY & SEROLOGY

FINALS L1: AUTOIMMUNITY PART 1

AUTOIMMUNITY
 It has already been established that the immune system is a
very potent and a strong system mainly programmed to CAUSES OF AUTOIMMUNITY
defend the body from infectious microbes or infectious  Genetics
agents that may cause harm. o Some diseases are associated with certain alleles
 It is also important to note that the immune system may also  Hormones
have collateral damages (like in a war) o Ex. HLA hormones can also play a role
 When an inflammatory reaction happens, there will be  Tissue trauma
casualties, such as normal tissue gets affected o Ex. induced in smoking
 There are instances where the immune system acts as a  Release of cryptic antigens
double-edged sword; it defends and attacks itself as the  Microbial infections
same time  Epigenetics
 When such things happen, this condition or group of
disorders are called Autoimmune diseases

ETIOLOGY OF AUTOIMMUNE DISEASE

SELF TOLERANCE
 Ability of the immune system to accept self-antigens and not
initiate a response against them
o The concept of immunology basically implies that
the immune system identifies self from non-self
 Non-self – attack
 Self – do not attack
 A type of immunologic tolerance
o A state of immune unresponsiveness that is
directed against a specific antigen, in this case, a
How does this factors interplay?
self-antigen.
 Ex. If a woman possesses hormones that can be a trigger to
 In order for self-tolerance to develop, lymphocytes must be
autoimmunity, plus also possessing HLA genes, there is a
“educated” so they can distinguish between self-antigens
higher possibility that it is easier for the woman to lose
and foreign antigens.
immunologic tolerance, the T-cells become self-reactive to
 This education takes place at two levels:
self-antigen, and will most likely develop immune diseases
o Central
o A lot of women are prone to immune diseases
o Peripheral.
 In the case of men, especially smokers, it may also cause a
trigger even if there is lesser susceptibility, and may still lose
CENTRAL TOLERANCE immunologic tolerance and proceed to autoimmune diseases
 Occurs in the central or primary lymphoid organs, the
thymus, and the bone marrow AUTOIMMUNE DISEASES
 In the thymus, there is a process known as negative
selection SYSTEMIC IMMUNE DISEASES
o T cells that express T-cell receptors (TCRs) with a  Affects the whole body
strong affinity for these selfantigens are deleted by  Organ-specific
apoptosis  Encounter anti-nuclear antibody
o Those T cells that have a receptor for the self-
antigen are eliminated SYSTEMIC LUPUS ERYTHEMATOSUS
 A chronic systemic inflammatory disease which appears to
 In the bone marrow, those with receptors having a strong originate from complex interactions between environmental
affinity for self-antigens are eliminated by apoptosis factors, genetic susceptibility, and abnormalities within the
 Some self-reactive B cells are not deleted: rather, they are immune system
stimulated to rearrange their immunoglobulin genes o Occurs when there is an inflammatory reaction in
(preparation to become a plasma cell) so that their B-cell the whole body and persists for a long period of
receptors are no longer antigen specific. time
o This process is called Receptor Editing o There are no bacteria, microbes, or tumor cells for
o The receptors undergo receptor editing where they the immune system to fight against; it is really just
are rearranged so that they no longer become war inside the body
antigen-specific  Women are much likely to be affected than men
 Peak age of onset: 20-40 years old
ANERGY
 B cells that possess receptors that only weakly recognize  There are a lot of autoantibodies associated with SLE
self-antigens are induced to downregulate the expression of o These include antibodies to double-stranded DNA
their receptors and develop a specific state of (dsDNA), histones, and other nuclear components,
unresponsiveness to the antigens as well as autoantibodies to lymphocytes,
 When the B cells has receptor for a self-antigen but is erythrocytes, platelets, phospholipids, ribosomal
unresponsive components, and endothelium.
PERIPHERAL TOLERANCE  Anti-dsDNA and complement proteins have been found in
 Lymphocytes that recognize self-antigens in the secondary immune complexes that are deposited in organs such as the
lymphoid organs are rendered incapable of reacting with kidneys and skin and are thought to play a major role in the
those antigens pathogenesis of SLE.
o Mechanism for lymphoid organs  The immune complexes in the kidney can trigger TYPE III
HYPERSENSITIVITY
 The accumulation of IgG to the dsDNA form complexes of an
intermediate size that becomes deposited in the glomerular
basement membrane of the kidney

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 This immune complexes also induce the generation of

chemotaxin such as C5a
o The activation of complement results in
recruitment of neutrophils, which release
lysosomal enzymes that cause injury to the
surrounding tissues, thereby leading to a
hypersensitivity reaction

 Over 100 autoantibodies associated with SLE have been

discovered
o A typical SLE patient would have an average of 3
out of the 100 autoantibodies present in the
system.

 Aside from antibodies that causes type 3 hypersensitivity,

other antibodies may also have consequences to the cells

CONSEQUENCES OF AUTOANTIBODIES TO CELLS DIAGNOSTIC CRITERIA

Antibodies to RBCs Hemolytic anemia  There are 11 clinical criteria, and 6 immunologic criteria
Antibodies to platelets Thrombocytopenia for the diagnosis of SLE
Antibodies to endothelial Vasculitis  The patient must satisfy 4 out of the 17, including at
cells Psychiatric problem least 1 clinical criterion and 1 immunologic criterion to
Miscarriage be classified as SLE
Stillbirth  Elevated antinuclear antibody titer
Phospholipid antibodies Pre-term delivery  Elevated anti-dsDNA titer
 Occurs in pregnant with lupus  Presence of antibody to the Sm nuclear antigen
Neonatal lupus  Presence of antiphospholipid antibody
 Occurs in up to 8% of babies born to  Low complement levels
pregnant women with SLE, is  Positive direct Coombs’ test in the absence of hemolytic
associated with antibodies to the anemia
SS-A/Ro nuclear antigens
SS-B/La Utero heart block LABORATORY DIAGNOSIS
 A serious complication that occurs in  When SLE is suspected, screening test is first done for the
2% of fetuses whose mothers have anti-nuclear antibodies
anti–SS-A antibodies o Present in majority of patients
 Leukopenia
CLINICAL SIGNS AND SYMPTOMS  Anemia
 Thrombocytopenia
 Elevated ESR with a normal/low CRP

ANTINUCLEAR ANTIBODIES (ANA)

Arthritis Erythematous rash Nephritis  Are heterogeneous group of antibodies that have different
antigen specificities
 The nuclear antigens they are directed against can include
the dsDNA, ssDNA, histones, nucleosomes (DNA histone
complexes), centromere proteins, and extractable nuclear
ARTHRITIS antigens
 The progression of lupus starts with symmetric arthritis
o Joint pain all over the body especially in the hands EXTRACTABLE NUCLEAR ANTIGENS (ENAs)
o Both hands will have the joint pain  Group of nuclear antigens that were so named because they
are isolated in saline extracts of mammalian tissues
ERYTHEMATOUS RASH  These antigens represent a family of nuclear proteins
 The anti-dsDNA forms immune complexes that get deposited associated with uridine-rich RNA ENAs
in the skin, which results to erythematous rash  The ENAs include:
 Also called macupapular rash, or “butterfly rash” o Ribonucleoproteins (RNP)
 Lupus – means “wolf” because of the wolfish appearance o Sm antigen
o SS-A/Ro and SS-B/La antigens
NEPHRITIS o Scl-70
 Last progression is the damage to the kidney, causing o Jo-1
nephritis o PM-1
 Complications may lead to death
Self-study guide: create a table listing the common ANA,
BUTTERFLY RASH OF SLE characteristic, immunofluorescent pattern, and disease association
 Characteristic rash over the cheekbones and forehead (located at the last page)
(diagnostic of SLE)
 Not all SLE patient may manifest this rash (60-70%) METHODS OF ANA DETECTION

INDIRECT IMMUNOFLUORESCENCE (IIF)

FLUORESCENT ANTINUCLEAR ANTIBODY (FANA)

 Most widely used and accepted test
 Highly sensitive, detects a wide range of antibodies, and is
inexpensive and easy to perform
 In addition, the antigens are in their original form and
location within the cells used in the test
 This test has several applications.

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 It has been used as a screening test to identify patients who  The homogenous pattern is found in patients with SLE, drug-
have ANAs as well as patients who are negative for ANAs to induced lupus, and many other autoimmune diseases
provide guidance in selection of follow-up assays based on
immunofluorescence patterns and to monitor ANA titers in
patients during treatment
o Screening test
o For Px who are ANA-negative
o Monitor ANA titers during treatment
 SCREENING TEST
o Commonly performed with a 1:40 or 1:80 dilution
of patient serum
 To avoid detecting low positive titers
seen in healthy persons

Titer of ≥ 160
 To make the test more specific PERIPHERAL (RIM / OUTLINE)
o  In this pattern, diffuse staining is seen throughout the
 Considered clinically significant nucleus, but there is a greater staining intensity around the
o Patient samples that are positive on the ANA outer circle surrounding the nucleus in interphase cells
screen are serially diluted and tested to determine  Dividing cells show strong staining of the condensed
the antibody titer, specified as the highest dilution chromatin
to show nuclear fluorescence.  This pattern is primarily caused by antibodies to dsDNA and
FLUORESCENT ANA TEST is highly specific for SLE.

SPECKLED
 This pattern is characterized by discrete, fluorescent specks
throughout the nuclei of interphase cells.
 Staining is absent in the nucleolus and in the chromatin
region in dividing cells.
o Specks of stars in the sky
 The speckled pattern is associated with antibodies to ENAs
and can be found in patients with SLE, Sjögren’s syndrome,
systemic sclerosis, and other systemic autoimmune
rheumatic diseases.

 Viewed under the microscope

 There is a commercially prepared microscope slide
 In the slide, there are nucleated cells
o Derived from the human epithelial cells lines: Hep-2
 Standard substrate for most clinical NUCLEOLAR
laboratories worldwide  Prominent staining of the nucleoli within the nuclei of
 Hep-2 have large nuclei and is easy to interphase cells
visualize, has high antigen expression,  The size, shape, and number of the nucleoli per cell are
high sensitivity, and facilitating variable and staining can be smooth, clumpy, or speckled,
visualization of result depending on the type of antibody present
 Incubate patient serum with the Hep-2 coated slide  Staining may or may not be present in the dividing cells.
 Wash to remove unbound antibodies  The nucleolar pattern is primarily caused by antibodies to
 Add anti-human immunoglobulin labeled with a fluorescent RNA and RNP and is seen mainly in patients with
tag to detect bound IgG (Total immunoglobulin) scleroderma, but can also be present in patients with other
o The tag is in the second antibody connective tissue diseases
 After incubation and washing, view under a fluorescent
microscope (preferred)
o May also be viewed under light microscope but with
modifications into the protocol

PATTERN OF IMMUNOFLUORESCENCE
 In addition to the antibody titer, the pattern of fluorescence is
also reported:
CENTROMERE
o It provide clues about the autoantibody present
 Smaller than speckled pattern
and associated diseases  Numerous discrete speckles are seen in the nuclei of
interphase cells and the chromatin of dividing cells.
HOMOGENOUS PATTERN  Most cells have 46 speckles, representing the number of
 This pattern is characterized by uniform staining of the entire
chromosomes.
nucleus in interphase cells and of the condensed  This pattern is caused by antibodies to proteins in the
chromosomal region in metaphase cells
centromeres of the chromosomes and is found mainly in
 The whole cell is glowing
patients with the CREST syndrome
 It is associated with antibodies to dsDNA (also known as
native or nDNA), histones, and deoxyribonuclear protein
(DNP)

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MIXED PATTERNS
 Mixed patterns can also be observed; in some cases, one
pattern may totally or partially obscure another (for example,
a homogeneous pattern might cover up a speckled pattern)
 In these cases, titration of the patient serum can help to
distinguish between the separate patterns and an antibody
titer would be reported for each one.
 If the FANA test is negative, no clearly discernable
fluorescent pattern is observed in the nuclei of the cells.
 Up to 5% of SLE patients test negative, so this test cannot
be used to absolutely rule out SLE
o If positive – diagnostic
o If negative – does not rule out SLE

ANTIPHOSPHOLIPID ANTIBODIES


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