Compiled Is Trans

IMMUNOLOGY AND
TRANSPLANT IMMUNOLOGY SEROLOGY (IS)

TUMOR IMMUNOLOGY / GROUP 2 / BSMT 2B
TRANSPLANT IMMUNOLOGY intestine) transplants were performed

 is the study of the immune in the United States.1 Today, more
response that occurs when an than 50,000 hematopoietic stem cell
organ or tissue is moved (grafted) (HSC) transplants are performed
from one individual to another. worldwide each year for a variety of
indications, including cancer,
autoimmune disease,
TRANSPLANTATION
immunodeficiencies, and other
 is a potentially lifesaving diseases.
treatment for end-stage organ Of critical importance has been the
failure, cancers, autoimmune growing knowledge of the
diseases, immune deficiencies, and immunologic mechanisms of graft
a variety of other diseases rejection and graft-versus-host
 is where you implant a “non-self” disease (GVHD), in particular the
tissue into the body. role of human leukocyte antigens
 is necessary because (HLAs) and the development of
when the recipient's organ has pharmacological agents that promote
failed or has been damaged by a graft survival by interfering with
disease or various components of the immune
injury, transplantation is one system.
of the great advances in modern The number of transplants performed
medicine. is a testament to the numerous
developments over the past few
NOTE: The first organ decades in patient management pre-
transplantation, using a kidney from and post-transplant and in the
an identical twin, was performed in technologies for organ or stem cell
1954 by Dr. Joseph Murray at Peter acquisition and sharing.
Bent Brigham Hospital in Boston.
The recipient survived for 9 years.
TERMINOLOGIES:
Dr. Murray was ultimately
recognized for his work by receiving
Donor
the Nobel Prize in Medicine in 1990.
 which is the individual who
At present, a variety of tissues and
provides the graft
organs are transplanted in human
beings, including bone marrow, Recipient or Host
peripheral stem cells, bone matrix,  which is the individual who receives
skin, kidneys, liver, cardiac valves, the graft.
heart, pancreas, corneas, and lungs
In 2015, 30,900 solid organ (kidney,
pancreas, liver, heart, lung, small
IMMUNOLOGY AND
Graft rejection
 involves immune reactivity of the Allogeneic MHC molecules on
recipient against transplanted transplanted tissue trigger strong
allografts. immune responses causing
rapid graft rejection, and
Graft-versus-host-disease (GVHD) immunosuppressive therapy is a
 is triggered by the reactivity of drug regimen that patients use to
donor-derived immune cells against lower their bodies' immune response.
allogeneic recipient tissues. These drugs help doctors stop the
immune system from overreacting
HISTOCOMPATIBILITY and damaging transplanted organs
and tissues. So, without it,
SYSTEM HLA System transplantation is more likely to
 is the strongest immunologic barrier result in a graft rejection that is why
to successful allogeneic organ and mostly everyone has to
HSC transplantation take immunosuppressant
 it consists of cell surface proteins drugs when receiving an organ
that play a central role in thymic transplant. Remember, alloantigens
education of T lymphocytes, that are encoded by the genes of the
initiation of adaptive immune MHC system are going to induce the
responses, and regulation of other strongest immune rejection thereby
immune system components. rejecting tissues.
 plays a critical role in regulating the The MHC gene products have an
immune response. As a consequence important role in clinical
of its role in immune regulation and immunology. For example,
exquisite polymorphism, the HLA transplants are rejected if performed
system also constitutes an against MHC barriers; thus,
immunological barrier which must immunosuppressive therapy is
be avoided or otherwise overcome required
in clinical transplantation.
NOTE: HLA proteins are found on the Major Histocompatibility Complex

surface of almost all nucleated cells and (MHC) Antigens
are antigenically very diverse in the  main function is to bring antigen in
human population. Therefore, the body to the surface of cells for
transplantation of a solid organ or HSCs recognition by T cells.
into an allogeneic host is likely to result in  The histocompatibility complex that
graft rejection in the absence of encodes cell surface antigens was
immunosuppressive therapy. first discovered in graft rejection
experiments with mice. When the
antigens were matched between
IMMUNOLOGY AND
donor and recipient, the ability of a
graft to survive was remarkably
improved.
The presence of HLA was first
Classical (transplant) Antigens recognized when multiply
 also known as major transfused patients experienced
histocompatibility complex (MHC) transfusion reactions despite
antigens proper crossmatching. It was
 are composed of the class I and class discovered that these reactions
II proteins. were caused by leukocyte
antibodies rather than by
Class I proteins include: antibodies directed against
 HLA-A, HLA-B, and HLA-C erythrocyte antigens. These same
antibodies were subsequently
 present endogenously synthesized
discovered in the sera of
antigens to CD8+ cytotoxic T cells
multiparous women.
 highest on lymphocytes and myeloid
cells and low or undetected on liver
hepatocytes, neural cells, muscle HLA genes
cells, and sperm  are inherited as haplotypes from
parental chromosomes
 expressed on all nucleated cells
Haplotype
Class II proteins consist of:  a group of closely linked alleles on a
 HLA-DR, HLA-DQ, and HLA-DP single chromosome;
 are found primarily on APCs that  for example, HLA-A1, HLA-Cw7,
include B lymphocytes, monocytes, HLA-B8, HLA-DR3, and HLA-
macrophages, dendritic cells, and DQ2.
thymic epithelium
Off- spring receive one maternal and
 are present exogenously synthesized
one paternal HLA haplotype.
antigens to CD4+ T helper cells
HLA Proteins
 are encoded by a set of closely
linked genes located on the short arm
of chromosome 6 within the MHC
region.
NOTE:
IMMUNOLOGY AND
TUMOR IMMUNOLOGY / GROUP 2 / BSMT 2B the entire MHC is inherited as
an HLA haplotype in a Mendelian
fashion from each parent. The
segregation of HLA haplotypes
HLA genes are closely linked and
within a family can be assigned by
family HLA studies.
HLA antigens are inherited in a
Mendelian dominant manner. HLA
genes are almost always inherited  have critical roles in the
together, thus the antigens of the development and functioning of the
entire HLA region inherited from innate and adaptive immune systems.
one parent collectively are called  serve as ligands for regulatory
haplotype. Because chromosome 6 is receptors on natural killer (NK) cells
an autosome (a chromosome with in the innate immune response.
two pairs), all individuals have two
HLA haplotypes (one for each
chromosome). NOTE:
The human leukocyte antigen (HLA)

Based on Mendelian inheritance, there system (the major histocompatibility
is a: complex [MHC] in humans) is an
a 25% chance that any two siblings important part of the immune system
will inherit the same two haplotypes and is controlled by genes located on
(i.e., are HLA identical), chromosome 6. It encodes cell
a 50% chance of them being HLA surface molecules specialized to
haploidentical (i.e., share one of two present antigenic peptides to the T-
HLA haplotypes), and; cell receptor (TCR) on T cells.
a 25% chance of them being HLA
nonidentical (i.e., share neither HLA HLA proteins have critical roles in
haplotype). the development and functioning of
the innate and adaptive immune
systems. They serve as recognition
HLA Proteins elements for antigen receptors on T
 heterodimeric molecules consisting lymphocytes, thus initiating adaptive
of two different polypeptide chains immune responses.
chemically bound to each other They also serve as ligands for
 also, members of the regulatory receptors on natural killer
immunoglobulin superfamily, which (NK) cells in the innate immune
share structural similarities with response; The CD8 molecule on
immunoglobulin molecules. cytotoxic T lymphocytes interacts
with class I HLA proteins, whereas
the CD4 molecule on the T helper
(Th) cell subset interacts with class II
HLA proteins.
Class I Proteins
 are the product of the HLA-A, HLA-
B, and HLA-C genes and are
IMMUNOLOGY AND
expressed on the cell surface
covalently bound to
β2–macroglobulin
 nucleated cells.
CLASS II PROTEINS
 are the products of the HLA-D
region genes
 antigen-presenting cells
Polymorphism
 refers to the presence of two or more
different genetic compositions
among individuals in a population.
HLA SYSTEM
 the most polymorphic genetic system
in humans
 Why is it the most polymorphic?
This was also already tackled in our
previous discussions, right? So, why
is it polymorphic? There are more
than 2,013 different alleles of HLA-
A, over 2,605 alleles of HLA-B, and
1,551 alleles of HLA-C that have
been identified at this time
 Why is this important? this
polymorphism is essential to our
survival because it allows for an
immune response to diverse
immunogens
Minor Histocompatibility Antigens

(mHAs)
 tissue rejection in MHC-identical
transplants and development of
GVHD in HLA-identical sibling
HSC transplants
IMMUNOLOGY AND
TUMOR IMMUNOLOGY / GROUP 2 / BSMT 2B  a “slower” rejection pace was
mediated by these
transplantation antigens
 are non-HLA proteins that
demonstrate variation in the amino
acid sequence between individuals
HLA match. Up to one third of

NOTE: transplants can be rejected because
of minor antigens. (Turgeon)
Researchers identified a second set Transplanting one individual’s tissue
of transplantation antigens based on or cells containing a polymorphic
studies in mice and humans. These variant of one of these proteins into
studies demonstrated tissue rejection another individual possessing a
in MHC-identical transplants and different polymorphic variant can
development of GVHD in HLA- induce a recipient’s immune
identical sibling HSC transplants.3 response to the donor variant. CD4
The scientists conducting these or CD8 T cells recognize the variant
studies also observed that a “slower” protein in an MHC-restricted fashion
and mediate the immune response.
rejection pace was mediated by these
This response is analogous to the
transplantation antigens, thus their reaction to a microbial antigen.
name—minor histocompatibility Several mHAs have been identified,
antigens (mHAs). including proteins encoded by the
Minor histocompatibility male Y chromosome, proteins for
antigen (also known as MiHA) are which the recipient has a
receptors on the cellular surface of homozygous gene deletion, proteins
donated organs that are known to that are autosomal encoded, and
give an immunological response in proteins that are encoded by
some organ transplants. They cause mitochondrial DNA.
problems of rejection less frequently
than those of the
major histocompatibility Major Histocompatibility Complex
complex (MHC). (MHC)
NOTE: A recipient may respond to  they are a group of complex
minor histocompatibility antigens. histocompatibility antigens which
Minor antigens are encoded by genes cause RAPID and STRONG
outside the HLA. These minor immunoreaction to the graft.
histocompatibility antigen
mismatches are not detected by Minor histocompatibility antigens
standard tissue typing techniques but  are a group of complex
may cause rejection despite a good histocompatibility antigens which
cause SLOW and WEAK
immunoreaction to the graft
MHC Class I-Related Chain A (MICA)
Antigens
 is polymorphic, with over 50 allelic
variants. MICA proteins are
expressed on endothelial cells,
IMMUNOLOGY AND
keratinocytes, fibroblasts, epithelial
cells, dendritic cells, and monocytes,
but they are not expressed on T or B
lymphocytes.
 As such, MICA proteins could serve  it is used to group human blood
as targets for allograft immune into different types based on the
presence or absence of certain
responses.
markers on the surface of RBCs.
 The four main blood types are A,
B, O and AB.
NOTE: ABO blood group incompatibility

 is a barrier to solid organ
Antibodies to MICA antigens have transplantation.
been detected in as many as 11% of  People with have only one blood
kidney-transplant patients and are type received blood from someone
associated with rejection episodes different blood type it may cause
and decreased graft survival. their immune system to react.
The association Hyperacute rejection
of antibodies against MICA and allograft  lead to rapid rejection of
rejection was highly significant in transplanted organ graft within
patients with no 24hrs of reperfusion.
HLA antibodies indicating MICA  Anti – A or anti- B antibodies
antibodies as an independent risk develop in individuals lacking the
factor in graft survival. This may corresponding blood group
explain for the allograft rejection in antigens. As such, recipient-donor
well-matched kidney donors with no pairs must be ABO identical or
HLA antibodies. compatible to avoid adverse
outcome.
 transplantation approaches using
ABO Blood Group Antigens plasma exchange and intravenous
immunoglobulin have allowed
ABO system successful transplantation of
 is the only blood group system that kidneys from ABO- incompatible
affects donor and this procedure reduce
ABO antibody to lower the risk of
Hyperacute rejection.
Clinical Transplantation Killer Immunoglobulin-Like Receptors

 aims to serve as a channel of rapid (KIRs)
communication for all those  one of the several types of cell
involved in the care of patients surface molecules that regulates
who require organ or tissue the activity of NK lymphocytes.
transplants.  it contains activating and
inhibitory receptors that vary in
IMMUNOLOGY AND
number and type on any

individual NK cells
 The balance of signals received by
the activating and inhibitory
receptors regulates the activity of
NK cells.
 the ligands for the inhibitory KIRs  is the ability of an individual
have been defined as several of the organism to distinguish its own
class I MHC molecules including tissues from those of another. It
HLA-, HLA-B, and HLA-C
manifests itself in the recognition of
proteins.
 The regulatory role of KIRs has antigens expressed on the surface of
been exploited in haploidentical cells of non-self-origin
stem cell transplantation – a type  In transplantation, the introduction of
of allogeneic transplant where ‘non-self’ cells or tissues into a
donor matches exactly half of your recipient can trigger an immune
HLA that uses healthy, blood
response.
forming cells from a half-matched
donor to replace the unhealthy Autograft
ones.  Is the transfer of tissue from one area
of the body to another of the same
In self antigens, it is the individual
convention antigens in the body of Syngeneic graft (also known as an
an individual. Isograft)
 humoral immune response to self-
 Is the transfer of cells or tissues
antigens in transplant recipients
have been associated with poor between individuals of the same
transplant outcomes. species who are genetically identical
(e.g., identical twins)
Proteins described post-transplantation Allograft (homograft)

are:  Is the transfer of cells or tissue
1. angiotensin II type 1 between two genetically disparate
receptor individuals of the same species.
2. K-alpha 1 tubulin  grafted donor tissue or organ
3. Collagen-V contains antigens not present in
4. Myosin
5. Vimentin recipient
Xenograft (heterograft)
 Is the transfer of tissue between two
ALLORECOGNITION
individuals of different species (e.g.,
pig heart valve to a human heart)
NOTE:
 Most transplants fall into the
category of allografts
IMMUNOLOGY AND
 The HLA (human leukocyte

antigen) disparity between donor
and recipient that occurs with
allografts and xenografts will result
in a cellular and humoral immune
response to foreign MHC antigens.
 An in vitro correlate of direct
allorecognition
TWO DISTINCT MECHANISMS  In this assay, lymphocytes from an
Direct allorecognition individual needing a transplant are
 Is the process by which donor- incubated with lymphocytes from a
derived major histocompatibility potential donor that have been
complex (MHC)-peptide complexes, inactivated so they cannot proliferate
typically presented by donor-derived  The difference between the two
‘passenger’ dendritic cells, are lymphocyte populations can be
recognized directly by recipient T quantitated by the incorporation of
cells. radio-labeled thymidine into the
 Recipient T-cells bind and respond DNA of the proliferating cells.
directly to foreign (allo) HLA  The high level of radioactivity
proteins on graft cells. indicates that the recipients T cell
 Foreign HLA proteins may mimic a response to different antigens on a
self-HLA + peptide complex because potential donor’s cell that would be
of similarities in the structure of the more likely to simulate graft
allo-HLA protein itself or to rejection
structural similarities of allo-HLA
protein + peptide.
Indirect allorecognition
 Evidence suggests that virus-specific
T-cells may be an important source  Indirect pathway may contribute to
of alloreactive cells. Characterized the process of chronic rejection
by a high frequency (up to 10%) of  Presentation of processed donor
HLA peptides bound to HLA class II
responding T cells compared in a
molecules to CD4+ lymphocytes.
typical T-cell response to foreign
The result is antibody formation
antigen.
directed against the donor graft
 Direct pathway plays a major role in
 Second pathway by which the
the early weeks after transplantation
immune system recognizes foreign
HLA proteins.
MIXED LYMPHOCYTE REACTION  Involves the uptake, processing, and
(MLR) presentation of foreign HLA proteins
by recipient APCs to recipient T
cells.
 Analogous to the normal mechanism
of recognition of foreign antigens
IMMUNOLOGY AND
 Play a
predominant role
in induction of alloantibody and
chronic rejection
Effector responses against transplanted Such antibodies have been

allogeneic tissue include: produced in the recipient prior to
 Direct cytotoxicity transplant. In some cases, these
 Delayed-type hypersensitivity preformed antibodies to these
antigens may be present as a result
responses
of blood transfusion, prior
 Antibody-mediated mechanisms transplantation, or exposure of a
Rejection episodes vary in time of pregnant woman to fetal antigens
of paternal origin.
occurrence and the effector mechanism
Binding of performed antibodies or
that is involved. also called cytotoxic antibodies to
the alloantigens activates the
complement cascade and clotting
HYPERACUTE REJECTION mechanisms and leads to thrombus
 occurs within a few minutes to a formation. Thrombus formation is
few hours of transplantation or a blood clot that forms in a vessel
after the vascular supply to the and remains there. So, the
transplanted organ is established. thrombus it can become
Hyperacute reactions are caused problematic because it can clog
entirely by the presence of our vasculature. The clogging or
preformed humoral antibodies in blockage that our thrombus creates
the host, which react with donor can result to our ischemia and our
tissue cellular antigens. necrosis is transplanted in our
tissue. If we recall from our
Note: this type of rejection is due to histopathology, ischemia is the
PREFORMED HUMORAL shortage of oxygen to our tissues
ANTIBODIES, we can say that and necrosis is the death of our
hyperacute rejection is an antibody- body tissue. So how does
mediated type of rejection. thrombosis cause ischemia and
necrosis? Well, since there is a
It is a result of destruction of the blockage in our vasculature, our
transplant by so-called preformed blood won’t be able to flow which
antibodies to incompatible MHC carry our oxygen and other
antigens and, in some cases, to nutrients to our transplanted tissue.
carbohydrates expressed on This then causes our shortage of
transplanted tissues. our oxygen and nutrients to our
Mediated by preformed antibody tissue aka ischemia, that can result
that reacts with donor vascular to our tissue death aka tissue
endothelium. necrosis.
It is seldom encountered in clinical
transplantation
Donor-receipt pairs are chosen to
be ABO identical or compatible
IMMUNOLOGY AND
and patients awaiting
transplantation are screened for the
presence of preformed HLA
the doses of the immunosuppressive
antibodies. Some individuals
drugs lowers.
possess very low levels of donor-
specific antibody in the NOTE:
pretransplant period. In these The distinction between humoral
cases, antibody-mediated rejection acute rejection and acute cellular
may take place over several days. rejection is important because
This is what you call now the treatments may differ. If the acute
rejection is antibody mediated,
accelerated rejection.
patients are treated with B-cell-
directed drugs like Rituxan and
ACUTE REJECTION plasmaphoresis. Acute rejection may
 Days to months after transplant. be reduced by immunosuppressive
 Can be mediated by a cellular therapy, for example, with
alloresponse or by donor-specific cyclosporine and other drugs.
antibody. In contrast to hyperacute Interstitial cellular infiltrates contain
rejection which is humoral- a predominance of CD8+ T cells as
mediated, our acute rejection can well as CD4 T cells and
be of mediated either through our macrophages. The CD8 mediates the
cells or our antibodies which is cytotoxic reactions in foreign MHC-
humoral. expressing cells, while the CD4 cells
 seen in a recipient who has not produce cytokines and induce
previously been sensitized to the delayed-type hypersensitivity
transplant. reactions.
Antibody may also be involved in
There are two types of acute rejection for acute graft rejection by binding to
solid organs: vessel walls and activating
complement. The antibody induce
1. Antibody mediated or humoral transmural necrosis and
acute rejection inflammation as opposed to the
 Humoral acute rejection usually occurs thrombosis typical of hyperacute
within the first 3 months following rejection
transplantation. In the case of kidney Diagnostic criteria include
transplant, it will manifest a sudden characteristic histological findings,
decline of kidney function. deposition of the complement protein
C4d in the peritubular capillaries,
2. Acute cellular rejection (sometimes
and detection of donor-specific HLA
abbreviated as acute rejection).
antibody.
 usually occurs 1–6 weeks to several
years following transplantation when
CHRONIC REJECTION
Duration: Months to year
IMMUNOLOGY AND
Caused by both antibody and cell-
mediated immunity occurs in
allograft transplantation
E.g., In kidney transplantation,
chronic rejection is characterized by
slow progressive renal failure. Alloantibody production likely
The recipient’s circulation, contributes to the development of
lymphatic drainage, expression of chronic rejection as well
MHC antigens to the graft (e.g., bone NOTE:
marrow and skin grafts are very Chronic Rejection is indirect
sensitive to rejection as compared to pathway of allo rejection that is T cells
heart, kidney and liver grafts). recognizing the donor cells and
Results from a process of graft activating and causing inflammation
arteriosclerosis characterized by and tissue destruction, said to be
progressive fibrosis and scarring indirectly activated against the
with narrowing of the vessel lumen HLA’s that are mismatched on the
donor cells. So, for example, the
caused by proliferation of smooth
kidney which is commonly donated
muscle cells.
organ. In kidney transplant, even if the
Remains the most significant cause organ is from your relatives like
of graft loss after the first-year post- father, daughter and so on. The
transplant because it is not readily 100% HLA’s match typically doesn’t
amenable to treatment. occur. So, what happens is that it will
Immunologic component: CD4 T have a mismatched HLA in the donor
cells and B cells. compared to the recipient that will
Cytokines and growth factors— lead to Chronic Rejection. So, how
secreted by endothelial cells, smooth chronic rejection occur? So, in the
span of years having donated kidney
muscle cells, and macrophages
cells, they will die and will be
activated by IFN gamma—stimulate
recycled. Also, the verdict cells will
smooth muscle cell accumulation in travel from the kidney to lymph
the graft vasculature. nodes and from that it eventually dies.
Donor cells die over time because
Notes: they are getting replenished, so many
of the dead cells go in the sites of host
FACTORS AFFECT THE
through phagocytosis. So, the peptide
DEVELOPMENT OF CHRONIC
of the HLA peptides will end up to the
REJECTION:
recipients, MHC class 2 molecules.
 Prolonged cold ischemia
And peptides are derived in the HLA
 Reperfusion injury
Molecules. If this occurs, CD4 will
 AR episodes appear at T cell receptor with an
 Toxicity from antigen binding site. This CD4 and T-
immunosuppressive drugs cell could recognize the h2 peptide
which are foreign or non-self that will
stimulate immune response against
the non-self-peptide, if the HLA are
mismatched.
IMMUNOLOGY AND
At the same time, B-cell will also
appear which is its binding site is
any mismatched foreign non self
HLA molecules, if recognized by a b 2 IMPORTANT PRINCIPLES HELP
cell, the process is peptide via TO EXPLAIN THE
receptor mediated endocytosis. So, PATHOPHYSIOLOGY OF ACUTE
the production of anti HLA GVHD
antibodies that can recognize donors,  It represents an exaggerated but
MHC doesn’t occur very quickly “normal” inflammatory response
after a tissue or organ donation, it against foreign antigens (the
will occur years by years. So now, ubiquitous host alloantigens)
majority of the organ transplant  Donor lymphocytes encounter
recipient will generate an anti HLA tissues in the recipient that have
antibody that will lead to a slow
often been profoundly damaged due
chronic rejection because it will
to the underlying disease of the host
cause a damage into the organ like
blood vessel damage or its or pre-HCT therapy regimens such
thickening. as radiation or chemotherapy.
IN HUMANS, GVHD MAY

PRODUCE:
 Splenomegaly (enlarged spleen)
GRAFT-VERSUS-HOST-
 Hepatomegaly (enlarged liver)
DISEASE (GVHD)  Lymphadenopathy (enlarged
Duration: Varies lymph nodes)
Can either be acute or chronic  Diarrhea
HSC transplants (and less commonly  Anemia
lung and liver transplants) are  Weight loss
complicated by a unique allogeneic  and other disorders underlying
response inflammation and destruction of
In this condition, lymphoid cells in tissues
the graft mount an immune response
against the host’s histocompatibility
antigens. GVHD is initiated by donor-
Acute graft-versus-host disease derived T cells that recognize the
(GVHD) occurs during the first 100 recipient’s MHC antigens (and
days post infusion and manifests in minor H antigen) as foreign.
the skin, gastrointestinal tract, and Standard procedure: is used to
liver eliminate virtually all mature T
cells from the donor hematopoietic
stem cell source
Cells that participate in the
destruction of host cells in GVHD:
IMMUNOLOGY AND
cytokines (mainly TNF-α and IL-
1)
Unless the reaction is controlled,
GVHD activates destructive
immune defense that may lead to  Suppress antigraft immune responses
the death of the recipients in solid-organ and HSC
Beyond 100 days post-transplant, transplantation is growing
patients may experience chronic  Used in several ways including
GVHD. This condition resembles induction and maintenance of
autoimmune disease, with fibrosis immune suppression and treatment
affecting the skin, eyes, mouth, of rejection.
and other mucosal surfaces  Combination of different agents are
NOTE: frequently used to prevent graft
Graft is a rare disease, it happens rejection.
as a complication of the bone  However, the immune suppressed
marrow or bone marrow state induced by these agents comes
transplantation, its equivalent is at a price of increased susceptibility
stem cell transplantation. How to infection, malignancies, and other
does it happen? The immune cells
of the donor recognize your body associated toxic side effects.
as a foreign tissue organ and reject
your body. So, what causes this? SEVERAL CLASSES OF
Example is the gender mismatch, IMMUNOSUPPRESSIVE AGENT:
the donor is a female whereas the
1. Corticosteroids
recipient is a male, so this causes
an increased risk of graft vs host  Potent anti-inflammatory and
disease. Also, the certain genetic immunosuppressive agents used for
matches, which is important in a immunosuppressive maintenance.
transplant.  At higher doses, they are used to
treat AR episodes.
Acute graft disease happen after  Decrease macrophage function and
months of transplantation usually
alter leukocyte trafficking patterns
presents with the involvement of
skin, the gastrointestinal tract or  Long term use is associated with
the liver. Once the disease several complications, including
becomes severe, it is hard to treat. hypertension and diabetes mellitus
 Reduce inflammation by inhibiting
macrophage cytokine secretion
IMMUNOSUPPRESSIVE AGENTS  directly inhibit antigen-driven T cell
proliferation, but steroids do not act
directly on the IL-2–producing T
cell. They do, however, inhibit
production of lymphokines by
preventing monocytes from releasing
IL-1, thereby blocking IL-
IMMUNOLOGY AND
1–dependent release of IL-2 from over many years. The body's
antigen-activated T cells. Other constant immune response against
activities of monocytes such as the new organ slowly damages the
inhibition of chemotaxis, are also transplanted tissues or organ.
likely to be important in the
immunosuppressive process
2. Antimetabolites
 Interfere with the maturation of
NOTES:
lymphocytes and kill proliferating
 Corticosteroids are often referred to
by the shortened term "steroids." cells
 Some corticosteroid medicines
include cortisone, prednisone and NOTES:
methylprednisolone. Prednisone is  Azathioprine was the first such agent
the most commonly used type of employed
steroid to treat certain rheumatologic  Replaced by mycophenolate mofetil,
diseases (like rheumatoid arthritis or which has a more selective effect on
lupus). lymphocytes.
 They lessen swelling, redness,
itching, and allergic reactions. They
 Mycophenolate is used in
are often used as part of the
combination with
treatment for a number of different
other medications to keep your body
diseases, such as severe allergies or
from attacking and rejecting your
skin problems, asthma, or arthritis
transplanted organ (such
as kidney, liver, heart). It works by
 Maintenance
immunosuppressive therapy is weakening your bod’s defense
system to help your body to accept
administered to almost all kidney
the new organ as if it were you own.
transplant recipients to help prevent
 Blocks lymphocyte proliferation by
acute rejection and the loss of the
inhibiting guanine nucleotide
renal allograft.
synthesis in lymphocytes
 Acute rejection may occur any time
from the first week after
3. Calcineurin inhibitors
the transplant to 3 months
 inhibiting the phosphatase
afterward. All recipients have some
calcineurin and then blocking
amount of acute rejection.
Chronic rejection can take place activation of the NFAT transcription
factor. Nuclear factor of activated T-
IMMUNOLOGY AND
cells (NFAT) is a family of
transcription factors shown to be
important in immune response. One
or more members of the NFAT
despite potential mismatches of the
family is expressed in most cells of
the immune system HLA system.
 This activity makes these drugs as a
 Tacrolimus (FK-506)
valuable agent in the chronic
- a macrolide with mechanisms
management of patients with
similar to that of cyclosporine, is
allografts.
derived from a fungus,
 Cyclosporine and FK-506
Streptomyces tsukubaensis found
(tacrolimus) are compounds that
in Japan.
block signal transduction in T
- 50 to 100 times more powerful
lymphocytes, resulting in impaired
than cyclosporine. Because FK-
synthesis of cytokines such as IL-
506 is a more potent
2,IL-3IL-4, and interferon-gamma.
immunosuppressant than
 Cyclosporine (Cyclosporin A)
cyclosporine, patient recovery
- isolated in 1971 from the fungus
time is faster. FK-506 has higher
Tolypocladium inflatum, has become
toxicity compared with
the mainstay of immunosuppressive
cyclosporine
therapy in transplantation.
- Patients receiving FK-506 have
- affects T cells preferentially by
increased susceptibility to
inhibiting the induction of cytotoxic
infections (e.g. CMV or
T cells. Unlike corticosteroids,
cytomegalovirus) and an
cyclosporine does not inhibit the
capacity of all accessory cells to increased risk of developing
release IL-1. lymphoma or post transplantation
- binds to cyclophilin and the lymphoproliferative diseases.
complex binds to and inhibits Inhibitors and inducers may
calcineurin (a protein phosphatase). demonstrate an altered rate of
This prevents activation of the IL-2 metabolism that requires an
transcription factor. adjustment in drug dose
- In pharmacologic doses, however,
 Rapamycin (sirolimus)
cyclosporin A does not grossly
- Structurally, sirolimus resembles
interfere with the activation and
tacrolimus and has the same
proliferation of suppressor T cells.
intracellular binding protein, but
Studies have shown prolonged renal
sirolimus has a novel mechanism
allograft survival with cyclosporin A,
of action
- also inhibits antibody production
- has been approved as an
adjunctive agent (in combination
IMMUNOLOGY AND
with steroids) for the prevention
of acute renal allograft rejection. have ever had a lung transplant or
The main side effects include liver transplant
increased risk of infections and  May cause your body to overproduce
lymphoma, white blood cells
hypercholesterolemia,  Also, it may increase the risk that
hypertriglyceridemia, interstitial you will develop an infection or
pneumonitis, insomnia and cancer, especially lymphoma or skin
tremor, and thrombocytopenia cancer
NOTES: 4. Monoclonal antibodies

 Calcineurin inhibitors are the most  Identical antibodies that are produced
effective immunosuppressive drugs from a single clone of plasma cells
in use  Found in individuals with multiple
 Calcineurin inhibitors are myeloma
used topically and systemically to  Monoclonal antibodies also
treat various inflammatory skin produced in industry by fusing an
diseases, especially: antigen-sensitized, splenic B
lymphocyte with non-secreting
 Atopic dermatitis - (eczema) is a myeloma cell, thus creating an
condition that makes your skin red immortal cell line that secretes an
and itchy. It's common in children antibody of a single idiotype.
but can occur at any age and it is  Bind to cell surface molecules on
long lasting (chronic) and tends to lymphocytes are used at the time of
flare periodically. It may be organ transplant and to treat severe
accompanied by asthma or hay fever. rejection episodes after
No cure has been found transplantation.
 Psoriasis- it is also a skin disorder  Basiliximab and daclizumab both
that causes skin cells to multiply up bind the CD25 receptor (IL-2
to 10 times faster than normal. This receptor) and thus interfere with IL-2
makes the skin build up into bumpy mediated T-cell activation.
red patches covered with white  Alemtuzumab used for induction
scales. therapy at the time transplantation.
 Sirolimus is also used in - Alemtuzumab is a humanized
combination with other medications anti-CD52 IgG1 monoclonal
to prevent rejection of kidney antibody that depletes CD52-
transplants Should not be use if you expressing cells from the
IMMUNOLOGY AND
TUMOR IMMUNOLOGY / GROUP 2 / BSMT 2B circulation. Robust clinical and
radiologic data, derived from
clinical trials and long-term
observational studies, indicate
that alemtuzumab induces a
marked immunosuppression
related to the depletion of
 Both are potent immunosuppressive
circulating T and B lymphocytes.
agents that deplete lymphocytes from
However, recent advances
suggest that the long-term the circulation.
clinical effects of alemtuzumab
are probably due to unique NOTES:
qualitative changes in the  Monoclonal and polyclonal
process of lymphocyte antibodies are important supporting
repopulation of the immune therapies
system. This leads to a particular  Both polyclonal and monoclonal
rebalancing of the immune antibodies are used in the prevention
system. and treatment of transplant rejection
NOTE:
 A problem with some monoclonal Clinical Histocompatibility Testing
antibody preparations is that the
patients can develop anti-mouse NOTES:
antibody that may interfere with the The goal of histocompatibility
effectiveness of these agents. testing is to ensure that a
transplanted organ remains viable in
5. Polyclonal antibodies the recipient for the longest period of
time.
 Used as induction agents or to treat
The human major histocompatibility
severe rejection
complex HLA is located on the short
 Thymoglobulin is an antithymocyte
arm of chromosome 6.
antibody prepared in rabbits and
Inherited as haplotypes (i.e., set of
ATGAM is a polyclonal antiserum
alleles) one from each parent.
prepared from the immunization of They are co-dominantly expressed
horses. HLA is extremely polymorphic. It is
- Generic name: lymphocyte one of the most polymorphic genetic
immune globulin system in man. Associated with each
- Brand name: ATGAM of the loci is a set of alleles, each of
which give rise to the production of a
unique antigenic specificity that is
expressed on cell surface and that
can be detected by specific
antibodies or immunologically
activated T cells.
A close match between a donor's and
a patient's HLA markers
IMMUNOLOGY AND
is essential for a successful transplant
outcome. HLA matching promotes the
growth and development of new The closer the HLA antigens on the
healthy blood cells (called transplanted organ match the
engraftment) and reduces the risk of a recipient, the more likely that the
post-transplant complication called recipient’s body will not reject the
graft-versus-host (GVHD) disease. transplant.
When performing an HLA typing
test for a kidney transplant, the
HLA (Human Leukocyte Antigen) following HLA antigens are looked
Typing at:
 Is the phenotypic or genotypic - HLA-A
identification of the HLA antigens or - HLA-B
genes in a transplant candidate or - HLA-DR
donor. Six HLA antigens are looked at for
Take note: each person
For clinical HLA testing, phenotypes Each person has two of each of the
or genotypes of classical transplant antigens (one inherited from the
antigens or genes are determined mother and one inherited from the
(HLA-A, HLA-B, HLA-Cw, HLA- father).
DR, HLA-DQ). By analyzing which six of these
This information used to find the HLA-antigens both the donor and
most suitable donor-recipient recipient have, scientists are able to
combination from an immunologic determine the closeness of tissue
standpoint. matching.
It must be stressed that others factors A six-antigen match is the best
must also be considered when compatibility between a donor and
choosing a particular donor for any recipient.
given patient, be it solid-organ or This match occurs 25% of the time
stem cell transplant. between siblings who have the same
mother and father.
For example: ABO compatibility and
infectious disease status are important
considerations in donor selection. HLA Phenotyping
The classic procedure for
In this test, donor’s antigens determining the HLA phenotype is
expressed on the surface of the (complement-dependent
leukocytes or their genes are cytotoxicity) CDC test.
matched with that of the recipient. Panels of antisera or monoclonal
antibodies that define individual or
groups of immunologically related
HLA antigens are incubated with
IMMUNOLOGY AND
lymphocytes from the person to be
HLA typed in separate wells of a
microtiter plate.
Each well of the plate contains
different antibody. A vital dye such as eosin red or
It is important to note that multisera trypan blue is added to
are used for HLA typing. distinguish live cells from dead
This requirement is based on the cells when they are viewed
presence of both unique epitopes on microscopically.
HLA molecules (those that define The dead cells, whose
the phenotypic specificity of a membranes have been more
specific HLA antigen) and permeable, are able to make up
Public epitopes (epitopes that are the dye and appeared colored,
present on more than one unique whereas the live cells, whose
HLA protein). membranes remain intact, cannot
Because responses to public epitopes take up the dye and remain
are common, many sera must be colorless.
used to define a pattern of reactivity
that correlates with a specific HLA The proportion of dead cells is estimated
antigen. by microscopic examination and scored
according the following scale, established
Take note: by the American Society for
T and B lymphocytes are used Histocompatibility and Genetics (ASHI):
for HLA class 1 typing, whereas 1= 0% to 10% cell death; negative
purified B lymphocytes are used 2= 11% to 20% cell death; doubtful
for HLA class II typing because negative
class II antigens are not found on 4= 21% to 50% cell death; weak
most T cells. positive
After incubation, complement is 6= 51% to 80% cell death; positive
added. 8= 81% to 100% cell death; strong
Binding of antibody occurs only positive
if the lymphocytes express the 0= unreadable
HLA antigen targeted by the
antisera. Take note:
A complement reagent derived The CDC method has several
from rabbit serum is added to each limitations for HLA typing.
well. If the cells possess the HLA Viable lymphocytes must be used,
antigen defined by antibody in that which demands timely performance
well, complement is activated and of the assay.
the cells are killed. Separation of T and B lymphocytes
is required for differentiation of class
I versus class II antigens.
IMMUNOLOGY AND
TUMOR IMMUNOLOGY / GROUP 2 / BSMT 2B In addition, the source of antisera for
HLA typing is not always consistent
or reliable.
Thus, reagents can vary in quality or
quantity over time.
Finally, the level of resolution (i.e.,
the ability to distinguish two closely
related yet distinct HLA antigens) is polysaccharide, teichoic acid, and
limited. peptidoglycan.
The limits of resolution don’t Lectin Pathway: Binding of
significantly affect the role of this mannose-binding lectin to mannose
technology for matching-solid organ residues on glycoproteins or
donors and recipients. carbohydrates on the surface of
However, for allogeneic stem cell microorganisms.
transplantation, a higher level of
resolution is required. DNA-based Complement-Dependent Cytotoxicity
(molecular) HLA typing methods are Test
now commonly employed in CDC is the mechanism by which
histocompatibility laboratories antibody-coated target cells recruit
because their higher resolution, and activate components of the
reagent quality, and amenability to complement cascade, leading to the
higher throughput formats overcome formation of a Membrane Attack
to the limitations of CDC based Complex (MAC) on the cell surface
methods. and subsequent cell lysis.
Complement system:
Collection of serum proteins HLA GENOTYPING
involved in lysis of cell membranes,  Molecular based HLA genotyping
mediation of inflammation, methods used polymerase chain
reaction (PCR) - based
enhancement of phagocytosis, and
amplification of HLA genes
metabolism of immune reflexes. followed by analysis of the
amplified DNA to identify the
Activation of Complement specific HLA allele or allele group.
Classical pathway: Immune
(antibody-antigen) complexes, PCR
require one IgM or IgG molecules  is a laboratory technique used to
Alternative Pathway: Antibody- copy and amplify small segments of
independent, microbial components DNA when there are no sufficient
suchaslipopolysaccharide, quantities for molecular and genetic
analysis.
when we say amplify, in layman’s

term it is to make a larger or greater
amount. So, we increase the segment
of DNA in PCR because the amount
IMMUNOLOGY AND
of either molecular or the genetic

analysis is insufficient.
So back to HLA genotyping, when
we say genotype, we are dealing
with the genes or our genetic
characteristics.
So, when the multiplication of HLA It is a rapid technique (3 hours) but
genes is done, it can proceed now on is relatively low resolution.
the analysis. So, the purpose of it is It is rapid but then in low
to identify the specific HLA allele or resolution however, this
allele group present in the amplified could be changed into a
DNA. higher resolution.
Higher resolution may be achieved
Three DNA based HLA typing methods at the cost of large numbers of
in use: PCRs, expense, and complexity.
AGAROSE GEL
1. Polymerase chain reaction with
sequence-specific PCR. (PCR-SSP)
 Most common approaches for
analysis involve PCR amplification
of HLA genes with panels of primer
pairs, each in which amplifies
specific alleles or related allele
groups. ELECTROPHORESIS= used to
 This uses probes specific for allelic detect amplification
variants of HLA genes, were the Agarose gel electrophoresis is
probes are specific for the allelic used to detect amplification
variant sequence and therefore only HLA GENOTYPE-> identified by
amplify that sequence. determining which primers
resulted in amplification.
So, in here, it uses a probe specific involves designing one or both
for the allelic variant of HLA primers so that they will or will not
genes which when recognized then allow amplification (the 3'-
it can be amplified. mismatch principle).
Those primer pairs that binds to
the target gene-> result to the
detection of the amplification NOTES:
product. This is the principle of a PCR-SSP,
It requires minimal amounts of so during this, multiple PCR
reaction is carried out which
DNA to start with.
specifically correspond to a
different variation of the gene or
what we called allele. So, this
happens because PCR SSP uses
multiple primers at the 3 prime
end.
As you can see in the illustration.
When an allele-specific product or
IMMUNOLOGY AND
a PCR product is present then the
PCR is successful and it is reliable. - cause by an A to T transversion in the
So, looking at the bases that are in sequence encoding codon 6 of the human
red color, the first one which is a T beta-globin gene.
match with A so there is
- so, for the diagnosis of this transversion
amplication. However, at the
as the speed PCR is often is used.
bottom, as you can see, they didn’t
- two sets of primers are used: one for the
match thus results now to nothing
normal allele (a base) and one for the
or no amplification.
sickle cell allele (t base)
RESULT: shows that the tested target
DNA contains the mutation that is
associated with the disease.
2. PCRsequence-specific
oligonucleotide probe
hybridization. (PCR-SSOP)
During this reaction control
 Second common approach for
primers are used. These primers
HLA genotyping.
amplify so called housing gene
that is present in all cells of an  Performing PCR-SSOP involves a
organism single PCR reaction that will
amplify all HLA gene variants at a
Example: beta globin- this reaction acts as specific locus.
an internal control during PCR to
distinguish between negative reactions NOTE: this is referred to as
and field reactions. generic amplification)
So, the difference of it from
An external positive control is also the PCR-SSP is that PCR-
used- this are usually DNA from SSOP uses a single PCR
patients of which the genotype is reaction to amplify all HLA
known. The reaction that is specific gene while SSP uses multiple
for this genotype should view the reaction.
appropriate PCR product.  Amplified gene-> hybridization
A negative control is also used, (with a panel of DNA probes, each
usually DNA substitute with water to specific for a unique HLA allele or
make sure none of the PCR allele group).
component are contaminated.  Labelled sequence-specific
This can be used in the diagnosis of: oligonucleotide probes specific for
Sickle cell anemia individual alleles are hybridized to
the immobilized DNA. The target
IMMUNOLOGY AND
DNA sequence is amplified by
PCR and immobilized on a filter.
= RESULT: those
specifically hybridize probes
to the amplified DNA will
only be detected.
The oligonucleotide probes
(18–24 nucleotides long)
carry a radioactive tracer, and
the pattern of binding with
the panel of probes identifies
the sequences present and
hence the genotype.
The technique is relatively
slow and requires a large
number of probes to cover all
the possible allelic variants
(e.g., 22 probes are required
for the DR52 family (DR3, 3. Sequence-based typing. (SBT)
DR5, and DR6) alone).  Third common type of HLA
If it is a previously genotyping
unrecognized allele, there  Involves a sequence of PCR-
will be no reaction, as no amplified HLA genes.
probe will be available.  The nucleotide sequence of the
Reverse SSOP can be HLA gene DNA is identified
performed by hybridizing directly.
target biotinylated DNA with  RNA is used as the original
immobilized oligonucleotide template to avoid amplifying
probes. pseudogenes.
This technique is faster and is  DNA is made initially by reverse
transcription.
more suitable for routine
Basically, from the name
diagnostic use.
alone we can say that this are
false genes. These are
unfunctional segment of a
gene that resemble a
functional gene.
 This is typically carried out using
SANGER DIDEOXY CHAIN

TERMINATOR SEQUENCING.
 Generic amplification of the HLA
genes of interest is conducted->
sequencing reaction using dideoxy
nucleotides.
IMMUNOLOGY AND
 The dideoxy terminator are Provide varying levels of resolutions

fluorescently labeled. that can be tailored to the specific
 AUTOMATEDDNA clinical need.
SEQUENCERS WITH So, in HLA genotyping there are
FLOURESCENT DETECTORS- levels of resolutions depending to the
detection of synthesized DNA clinical need.
molecule. DNA-BASED TYPING- provides
 The sequence of the target gene is results at a level of resolution
compared with an HLA sequence comparable to CDC-based typing
data base to determine the specific (antigen equivalent) or can provide
HLA allele for the patient. results at the allele levels that is
 SBT is the “GOLD STANDARD” required for matching of unrelated
that can detect new allelic variants HSC donors and recipients.
because it interrogates all So, it is recommended that HLA
nucleotides in the amplified target genotyping must be use for matching
region as opposed to PCR-SSP of unrelated HSC donor and
and PCR-SSOP that targets small recipients.
Allele levels HLA typing has
stretches of previously defined
demonstrated the incredible extent of
nucleotides.
polymorphism within the HLA loci.
So, SBT interacts to all
nucleotides resulting to the Remember, polymorphism is the
detection of new alleles. ability to create several different
It will identify previously forms or types among the members
unknown alleles and has of a single gene
revealed a degree of
heterogeneity within the
Other techniques:
HLA genes that had not
previously been recognized.
1. Reference-strand-mediated
 This technique is fast (16–24
conformation analysis (RSCA)
hours) and very accurate.
 is a conformational method that
offers high resolution.
HLA genotyping overcomes the  The HLA type is assigned based
limitations of CDC-based HLA on accurate measurement of
phenotyping. conformation-dependent DNA
Cells do not need to be viable in mobility in gel electrophoresis.
order to obtain DNA for HLA  This allows discrimination of HLA
typing. alleles that differ by one
Typing Agents are chemically nucleotide.
synthesized, (thus there is no reliance
of human donors of antisera)
IMMUNOLOGY AND
2. Variable N-terminal repeats
analysis (VNTR)
 looks for polymorphisms in the
non-coding repeated DNA and can
be used for detecting and HLA ANTIBODY SCREENING,
monitoring micro chimerism post- IDENTIFICATION, AND
transplant. CROSSMATCHING
 Fluorescently labelled PCR
primers amplifying short tandem Crossmatch test
repeat loci are employed to obtain  Antibodies to HLA antigens can be
a ‘VNTR profile’ for patient and detected in candidates and
donor to assess the chimeric status recipients of solid-organ
of the patient. transplants.
 The match between a recipient and
potential graft donor is assessed by
NOMENCLATURE FOR HLA typing blood-group antigens and
MHC antigens, and evaluating
TYPING
existing anti-donor antibodies
introns (cross-matching).
In addition, a negative crossmatch
 is any nucleotide sequence within
means there is no recipient antibody
a gene that is removed by RNA
that is reactive with donors cells or
splicing during maturation
graft and a positive cross match
portends to severe rejection if the
donor organ is transplanted. So it is
better if negative.
Trivia:
The first human kidney transplant, was
attempted in 1935 by a Russian surgeon.
It failed because a mismatch of blood
types between donor and recipient
caused almost immediate rejection of the
kidney.
in 1954 a team in Boston headed by
Joseph Murray performed the first
successful human kidney transplant
between identical twins, followed 3
years later by the first transplant between
nonidentical individuals.
These antibodies can develop in

response to:
1. multiple blood transfusions
2. prior HLA-mismatched transplants.
IMMUNOLOGY AND
3. produced by women who have had
multiple pregnancies in response to
paternally derived
fetal antigens.
to assess the effectiveness of therapy
Because of the potential adverse impact for antibody-mediated rejection.
HLA antibodies can have on graft The purpose of the test in post
survival. transplantation is mainly focus on
the prevention of antibody mediated
HLA antibody screen rejection
 through these patients awaiting solid-
organ transplantation are screened
periodically for presence antibodies. Crossmatching- performed before
 If detected, the HLA specificity of transplant to confirm the absence of
the antibodies is then determined so donor-specific antibody.
that donors possessing those HLA
antigens can be eliminated from The CDC method or the complement
consideration for donation to that dependent cytotoxicity is used for HLA
patient. typing that is also used for:
HLA antibody detection and
So, they are continuously tested or screen identification.
until a recipient received a donor organ In this case, panels of
that is compatible to without adverse lymphocytes with defined
effect. HLA phenotypes are
incubated with the patient’s
Antibody screening and serum.
identification If the serum contains HLA
 performed post-transplantation. antibodies, they will bind to
Post transplantation is after the those lymphocytes in the
operation. panel that express the
corresponding HLA antigen.
Purpose: Binding is detected by
Aid in the diagnosis of antibody- addition of a complement
mediated rejection reagent derived from rabbit
Even though there are test performed serum and a vital dye to
before, there is a need for a post assess cell death
transplantation test. This is the microscopically.
crucial part of every transplantation,
because this is where we get to prove the level of antibody in a serum sample
if the procedure is successful to the may be below the level detectable by the
transplantation. CDC assay.
anti-human globulin (AHG) can be
added to the CDC assay to increase
the test’s sensitivity.
AHG-CDC assay- detect lower
levels of antibody as well as isotypes
of bound antibody that do not
IMMUNOLOGY AND
activate complement and thus
wouldn’t normally be detected in the
the most common type of or organ to
standard CDC assay.
be transplanted is the kidney and it is
30 to 60 unique lymphocyte
considered to be the most successful
preparations are included in the
among other organ transplantation.
panel.
but then failures are mainly due to
percent panel reactive antibody
chronic rejection, nephrotoxicity of
(%PRA) - the proportion of
the calcineurin inhibitor agents and
lymphocytes in the panel that are
recurrent disease.
killed by the patient’s serum.
Liver and heart transplant have also
Note: provided excellent treatment. The
clinical observation is that livers are
the specificity of the antibodies can be
more tolerogenic than other solid
determined by evaluating the phenotype
organ allografts, but the basis for this
of the panel cells.
has not been clearly established.
calculated PRA (cPRA)

 is determined for organ ANTIBODY DETECTION AND
allocation. IDENTIFICATION
 In this approach, the HLA
antigens to which a candidate has Enzyme-linked immunosorbent assay
HLA antibody are determined (ELISA)
using solid-phase assays.  substitute for CDC-based HLA
 These antigens are then classified antibody testing.
as unacceptable antigens for  utilize purified HLA antigens bound
that candidate. to the wells of microtiter plates.
 donors expressing those antigens Patient serum is added to the wells of
are excluded from donation for the plate;
that candidate.  if HLA-specific antibody is
present, it will bind.
cPRA value (Bound antibody is detected by the
 proportion of potential donors in the addition of an enzyme-labeled anti-
donor pool possessing one or more immunoglobulin reagent.)
of the unacceptable antigens.  Wells of the ELISA plate may
contain a pool of HLA antigens-
For example, a recipient with an serves as a qualitative screen for the
HLA-A2 antibody would have a presence of HLA antibody in a
cPRA value of 47% if 47% of serum.
potential donors are projected to be  Alternatively, each well may contain
HLA-A2 expressing, based on HLA antigens representing a single
historic HLA typing data. donor and thus can be used in a
fashion analogous to a CDC-based
NOTE: analysis, allowing %PRA and
specificity to be determined.
IMMUNOLOGY AND
flow cytometry
 single cell analysis by flow
cytometry is the most sensitive
method for crossmatching and
antibody identification. multiplex bead array system
 Antibody in patient serum can be  A more recent version of flow
incubated with beads that are cytometry-based antibody detection
coated with purified HLA  can assess binding of patient
antigens, either from: antibodies to up to 100 different
o a pool of donors HLA antigens in a single tube using
o an individual donor a dedicated flow-based detection
o or a single purified or system such as a Luminex bead
recombinant HLA protein. array.
 Beads coated with pooled HLA
proteins are a sensitive qualitative Note:
screen for the presence of HLA Flow cytometry-based methods are
antibody because they will detect the most sensitive technology for
antibodies to most common HLA detecting HLA antibodies.
antigens.
provide the most specific
 Beads coated with purified HLA
determination of the specificity of
proteins from individual donors or
HLA antibodies when beads coated
with a single HLA type (referred
with a single HLA antigenic type are
to as single-antigen beads) are
used.
used to determine the specificity of
the HLA antibodies in a patient’s
serum; (this information is used to donor–recipient crossmatch test
determine the cPRA.)
 is performed once a suitable donor
 Patient serum is incubated with the
has been identified for a particular
beads and bound antibody is
patient. To confirm the absence of
detected by adding a FITC-labeled
donor-specific antibody.
anti-IgG reagent.
 Donor T and B lymphocytes are
incubated with recipient serum in a
CDC assay.
 this is done to allow the formation of
antigen-antibody complexes on the
cell surface.
 The unbound proteins are washed
away and the bound antibodies are
detected with a second antibody ,
anti-human immunoglobulin G(IgG)
labelled with chromophore.
Microscopic analysis
IMMUNOLOGY AND
 used to verify a lack of binding after
the addition of complement and a
vital dye to differentiate live from
dead cells.
Cell death
 an indication of recipient antibody
binding to donor HLA antigen(s).
flow cytometry using an FITC labeled

anti-IgG reagent
 binding of antibody can be detected.
flow cytometric crossmatch

 for antibody screening and
identification and the most sensitive
method for detecting donor-specific
antibody.
GROUP 2
Antonio, Kairylle Zhailee Gaile C.
Ignacio, Keejan Mari R.
Garcia, Ma. Eliz Angela B
Agonoy, Mark Angelo C
Salvador, Raven Denvert Pulido
Tarnate, Best Champ Balitnang
Alonzo, Adrian Dave
Gatan, Micah Mae S.
IMMUNOLOGY
TRANSPLANT IMMUNOLOGY AND
SEROLOGY (IS)
TUMOR IMMUNOLOGY / GROUP 2 / BSMT 2B Mosby.
Welsh, K. (1999). Molecular typing for the

MHC with PCR-SSP. PubMed.
https://pubmed.ncbi.nlm.nih.gov/112
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supplements/corticosteroid-oral- g-4068-2108/mycophenolate-mofetil-
route-parenteral- oral/mycophenolate-mofetil-
route/description/drg- oral/details
20070491#:~:text=Corticosteroids
% Sasazuki et al. (n.d.). Haploidentical
20(cortisone%2Dlike Transplant
%20medicines), https://bethematch.org/patients-and-
families/about-transplant/what-is-a-bone-
marrow-transplant/haploidentical-transplant/
(n.d.). Retrieved from

https://www.ncbi.nlm.nih.gov/books/NBK5
58995/#:~:text=They%20are%20primarily%
20used%20as,management%20of%20patien
ts%20with%20allografts.
BMC. (n.d.). Retrieved from
https://msddjournal.biomedcentral.com/artic
les/10.1186/s40893-017-
00244#:~:text=Robust%20clinical%
20and%20radiologic%20data,circula
ting%20T%20and%20B%20lympho
scytes.
1 Chapter 19: Immunodeficiency Diseases
Group 4:
Immunodeficiency Diseases
DEFECTS OF HUMORAL
IMMUNITY
SOURCE: KAPLAN
The molecular defects, signs, and symptoms

associated with defects of pllagocytic celis,
complement, and Band T cells.
[BSMT 2B- 2020-2021- Group 4] Page 1

DEFECTS OF PHAGOCYTIC CELLS DEFECTS OF T LYMPHOCYTES

AND SEVERE COMBINED
IMMUNODEFICIENCIES
● Patients with defects in B lymphocytes can

deal with many pathogens adequately, defects
in T lymphocytes are observed globally
throughout the immune system.
● Because of the central role of T cells in
activation, proliferation, differentiation, and
modulation of virtually
❏ All naturally occurring immune
responses, abnormalities in these cell
lines send shock waves throughout the
system.
❏ It is often a Herculean clinical effort to
dissect the cause-and-effect
relationships in such inherited diseases,
and their diagnosis is often one of trial-
and-error, which takes years to unravel.
❏ Although in some cases both B- and T-
lymphocyte defects may occur, the
initial manifestation of these diseases is
almost always infection with agents
DEFICIENCIES OF COMPLEMENT such as fungi and viruses that are
OR ITS REGULATION normally destroyed by T-cell-mediated
immunity.
The B-cell defect, if any, is usually not detected
for the first few months of life
because of the passive transfer of
immunologlobulins from the mother
through the placenta or colostrum.
The immune system is so compromised
that even attenuated vaccine
preparations can cause infection and
disease.
[BSMT 2B- 2020-2021- Group 4] Page 2

B- and T-Cell Deficiencies
[BSMT 2B- 2020-2021- Group 4] Page 3

Immunodeficiency The PIDs can affect one or more parts of the

immune system,
Diseases depending on the specific disease.
 primary effect on B cells and humoral
immunity,
 cell-mediated branch of the adaptive
immune system.
CHAPTER OUTLINE  components of the innate defense system
(phagocytic cells, complement, or NK cells.)
Clinical Effects Of Primary Figure 19–1 illustrates points in the development
Immunodeficiencies
of the
The Nine Categories Of Primary immune system at which some PIDs exert their
Immunodeficiencies
main effects.
Laboratory Evaluation Of Immune
Dysfunction
IMMUNODEFICIENCIES:
Are disorders in which a part of the body’s
immune system is missing or dysfunctional.
2 types of ID:
ᴥ Primary and Secondary ID
 People with these conditions have a
o decreased ability to defend
themselves against infectious
organisms and
are more susceptible to developing
certain types of cancer
 can be inherited or acquired secondary to
other conditions such as
certain infections, malignancies,
autoimmune disorders, and
immunosuppressive therapies.
 An example of a secondary
immunodeficiency is the acquired
immunodeficiency syndrome (AIDS),
which is caused by the human
immunodeficiency virus (HIV).
 Primary immunodeficiencies (PIDs), which
are inherited dysfunctions of the immune
system.
X-linked inheritance and, therefore,
affect primarily males.
 autosomal recessive or autosomal
dominant inheritance. In general, defects in humoral immunity
CLINICAL EFFECTS OF PRIMARY (antibody production)
IMMUNODEFICIENCIES
[BSMT 2B- 2020-2021- Group 4] Page 4

 result in pyogenic (i.e., pus-forming) in neutrophil function are usually reflected

bacterial infections, particularly of the in recurrent pyogenic bacterial infections or
upper and lower respiratory tract. impaired wound healing.
 Recurrent sinusitis and otitis media (i.e., ear  Abnormalities in macrophage function will
infections) arecommon. have effects on both the innate and the
 The clinical course of viral infections in adaptive defenses because macrophages
patients with predominantly antibody are involved in the nonspecific phagocytosis
deficiencies is not significantly different of microorganisms during inflammation as
from that in normal hosts, with the well as in the processing of antigens and
exception of hepatitis B, which may have a their presentation to T cells in humoral and
fulminant course in patients with cell-mediated immune responses.
o agammaglobulinemias, conditions
in which antibody levels in the blood o Reduction in the macrophage
are significantly decreased. population by splenectomy is
Defects in T-cell–mediated immunity associated with an increased risk of
result in recurrent infections with overwhelming bacterial infection
intracellular pathogens accompanied by septicemia.
o such as viruses, fungi, and The complement system is activated directly by
intracellular bacteria. antigens or by antigen–antibody complexes to
Congenital T-cell deficiencies almost produce biologically active molecules that enhance
always develop mucocutaneous inflammation and promote lysis of
candidiasis, a yeast infection that involves microorganisms.
the skin, nails, and mucous membranes. Deficiencies of complement components result
o They are also prone to disseminated  in recurrent bacterial infections and
viral infections, especially with autoimmune-type manifestations. The
latent viruses such as herpes severity of the conditions varies with the
simplex, varicella zoster, and particular complement component that is
cytomegalovirus. deficient.
Because T cells also play an important role  the components of the immune system
in tumor immunity, patients with these interact extensively through many
conditions are more susceptible to regulatory and effector networks, a defect
developing certain types of cancer. Age- in one branch of the system may affect
adjusted rates of malignancy in patients with other aspects of immune function as well.
immunodeficiency diseaseare 10 to 200 times  In many cases, it appears that deficiency of
greater than those observed in immunocompetent one component of the immune system is
individuals. accompanied by hyperactivity of other
Most of the malignancies are lymphoid and components.
may be related to persistent stimulation of o This may occur because persistent
the remaining immune cells, coupled with infections continuously stimulate
defective immune regulation. the available immune cells or
because a compensatory
Defects in other components of the immune mechanism has been activated to
system correct for the deficient immune
ᴥ Neutrophils ( first line of defense), defects function.
[BSMT 2B- 2020-2021- Group 4] Page 5

The deficiency may involve a component that In the past, the immunodeficiencies have been
normally broadly classified as defects in T cells, B cells,
exerts regulatory control over other components of phagocytes, complement proteins, and other
the immune system—control that is lacking in the components of the innate immune system. In 2014,
deficiency state. the International Union of Immunologic
 For instance, T helper (Th2) cells secrete Societies (IUIS) updated their classification of PIDs
cytokines that regulate the development of by grouping them into nine different categories
B cells into plasma cells. A defect in Th2 cell based on their characteristic clinical features,
function, such as a deficiency in CD40L (a immunologic defects, and genetic abnormalities.
molecule involved in binding to cell
receptors during T-dependent immune THE NINE CATEGORIES ARE:
responses), removes or creates an Category 1 Combined
imbalance in the regulation of those Immunodeficiencies
immune responses. Category 2 Combined
 Whatever the mechanism, many partial Immunodeficiencies
immunodeficiency states are associated With Associated or
with allergic or autoimmune Syndromic Features
manifestations, currently referred to as Category 3 Predominantly
autoinflammatory disorders
Antibody Deficiencies
Category 4 Diseases of Immune
Dysregulation
Category 5 Congenital Defects of
Phagocyte Number,
Function, or Both
Category 6 Defects in Innate
Immunity
Category 7 Autoinflammatory
Disorders
Category 8 Complement
Deficiencies
Category 9 Phenocopies of Primary
Immunodeficiencies
*Although the PID diseases are separated into
these categories, some diseases are listed in more
than one category because they possess
overlapping features.
 Category 3, Predominantly Antibody
Deficiencies, is discussed first because the
conditions in this category are the most
commonimmunodeficiencies,
representing about 50% of the PIDs.
THE NINE CATEGORIES OF PRIMARY
CATEGORY 3: PREDOMINANTLY ANTIBODY
IMMUNODEFICIENCIES
DEFICIENCIES
[BSMT 2B- 2020-2021- Group 4] Page 6

 This category encompasses conditions in in response to environmental stimuli.

which the main characteristic is low levels  IgM reaches normal adult levels first,
of serum immunoglobulins. around 1 year of age, followed by IgG at
 Immunoglobulins migrate in the “gamma about 5 to 6 years of age.
region” of the serum protein  In some normal children, IgA levels do not
electrophoreticprofileTherefore, reach normal adult values until
deficiencies of immunoglobulins have been adolescence.
termed agammaglobulinemias. Therefore, it is important to compare a child’s
The mechanisms of the agammaglobulinemias immunoglobulin levels to age-matched reference
ranges.
Transient Hypogammaglobulinemia of
Infancy With Normal Numbers of B
Cells
 All infants experience low levels of
immunoglobulins at approximately 5 to 6
months of age;
o however, in some babies the low
levels persist for a longer time.
 Because these children do not begin
synthesizing immunoglobulins promptly,
they can experience
o severe pyogenic sinopulmonary and
skin infections as protective maternal
IgG is cleared.
 Cell- mediated immunity is normal and there
may be normal levels of IgA and IgM.
 IgG appears to be the most affected,
include genetic defects in B-cell maturation dropping to at least 2 standard deviations
or mutations leading to defective (SDs) below the age-adjusted mean with or
interactions between B and T cells. without a depression of IgM and IgA.
 Immunoglobulin levels in infants with this
condition usually normalize spontaneously,
In evaluating immunoglobulin deficiency
often by 9 to 15 months of age.
states, it is important to remember that
The mechanism of this transient
blood levels of immunoglobulins change hypogammaglobulinemia is not known.
with age.
These patients have normal numbers of
The blood level of IgG at birth is about the circulating CD19+ B cells.
same as the adult level, reflecting transfer This condition does not appear to be X-
of maternal IgG across theplacenta. linked, although it is more common in
The IgG level declines over the first 6 males.
months of life as maternal antibody is The cause may be related to a delayed
catabolized. maturation of one or more components of
Levels of IgA and IgM are very low at birth. the immune system, possibly Th cells.
The concentrations of all immunoglobulins
gradually rise when the infant begins to X-Linked Bruton’s Tyrosine Kinase
produce antibodies at a few months of age (Btk) Deficiency
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X-linked hypogammaglobulinemia
 results from arrested differentiation at the
pre–B-cell stage, leading to a complete
absence of B cells and plasma cells.
 The underlying genetic mechanism is a
deficiency of an enzyme called the Btk in
B-cell progenitor cells.
 Lack of the enzyme apparently causes a
failure of immunoglobulin VH gene
Bruton’s tyrosine kinase (Btk) deficiency first rearrangement.
described in 1952, is X chromosome linked (affects o The syndrome can be effectively
males) treated by administration of
lack circulating mature CD19+ B cells and intramuscular or intravenous
exhibit a deficiency or lack of immunoglobulin preparations and
immunoglobulins of all classes.1,7,8 vigorous antimicrobial treatment of
they have no plasma cells in their lymphoid infections.
tissues.  The syndrome can be differentiated from
The patients do, however, have pre-B cells transient hypogammaglobulinemia of
in their bone marrow. infancy by the absence of CD19+ B cells in
Because of the lack of B cells, the peripheral blood, the abnormal
the tonsils and adenoids are small or histology of lymphoid tissues, and its
entirely absent and persistence beyond 2 years of age.
lymph nodes lack normal germinal  Immunologists have also described patients
centers. with a similar clinical presentation to Btk
T cells are normal in number and function. who have a genetic defect that is inherited
About half of the patients have a family in an autosomal recessive manner.
history of the syndrome.
They develop recurrent bacterial infections Selective IgA Deficiency
beginning in infancy as maternal antibody is Selective IgA deficiency is the most common
cleared. congenital immunodeficiency, occurring in about
The patients most commonly develop 1 in 500 persons of American or European descent.
sinopulmonary infections caused by  Most patients with a deficiency of IgA are
encapsulated organisms such as asymptomatic.
streptococci,meningococci, and  Those with symptoms usually have
Haemophilus influenzae. infections of the respiratory and
Other infections include bacterial otitis gastrointestinal tract and an increased
media, bronchitis, pneumonia, meningitis, tendency to develop autoimmune diseases
and dermatitis. such as
Some patients also have a susceptibility to o systemic lupus erythematosus
certain types of viral infections, including (SLE), rheumatoid arthritis (RA),
vaccine associated poliomyelitis. celiac disease, and thyroiditis.
In general, live virus vaccines should not  Allergic disorders and malignancy are also
be administered to immunodeficient more common.
patients.  About 20% of the IgA-deficient patients
[BSMT 2B- 2020-2021- Group 4] Page 8

who develop infections also have an IgG2 heterogeneous group of disorders with a
subclass deficiency. prevalence of about 1 in 25,000.
If the serum IgA is lower than 5  Although this is a low incidence, it does
mg/mL, the deficiency is considered make CVI the most common PID with a
severe. severe clinical syndrome
If the IgA level is two SDs below the  The disorder can be congenital or acquired,
age-adjusted mean but greater than or familial or sporadic, and it occurs with
50 mg/dL, the deficiency is partial. equal frequency in men and women.
 Lack of IgA is caused by impaired  CVI is characterized by
differentiation of lymphocytes to become hypogammaglobulinemia that leads to
IgA-producing plasma cells. recurrent bacterial infections, particularly
 IgE antibodies specifically directed against sinusitis and pneumonia.
IgA are produced by 30% to 40% of patients  In addition, up to 20% of CVI patients
with severe IgA deficiency. develop herpes zoster (shingles), a much
 These antibodies can cause anaphylactic higher incidence than in immunologically
reactions when blood products containing normal young adults.
IgA are transfused.  There is usually a deficiency of both IgA
severe IgA deficiency have no other and IgG, but selective IgG deficiency may
symptoms, the IgA deficiency may occur. CVI is often associated with a
not be detected until the patient spruelike syndrome characterized by
experiences a transfusion reaction, malabsorption and diarrhea. CVI is also
resulting in the production of associated with an increased risk of
anti-IgA antibodies. lymphoproliferative disorders, gastric
 Therefore, products for transfusion to carcinomas, and autoimmune disorders.
known IgA-deficient patients should be  The most common autoimmune
collected from IgAdeficient donors or manifestations of CVI are immune
cellular products should be washed to thrombocytopenia and autoimmune
remove as much donor plasma as possible. hemolytic anemia.
 Most gamma globulin preparations contain  Other symptoms may include
significant amounts of IgA. However, lymphadenopathy, splenomegaly, and
replacement IgA therapy is not useful intestinal hyperplasia.
because the half-life of IgA is short (around  CVI is diagnosed by demonstrating a low
7 days) and intravenously or intramuscularly serum IgG level in patients with recurrent
administered IgA is not transported to its bacterial infections.
normal site of secretion at mucosal  Additionally, blood group
surfaces. isohemagglutinins, or the so-called
 Furthermore,administration of IgA- naturally occurring antibodies, are typically
containing products can induce the absent or low.
development of anti-IgA antibodies or  In contrast to X-linked
provoke anaphylaxis in patients who agammaglobulinemia, most patients with
already have antibodies. CVI have normal numbers of mature B
cells. However, these B cells do not
Common Variable Immunodeficiency differentiate normally into
(CVI) immunoglobulin-producing plasma cells.
Common variable immunodeficiency (CVI) is a
[BSMT 2B- 2020-2021- Group 4] Page 9
 Three major types of cellular defects have normal range.

been  In patients with recurrent infections, levels
o T cells or their products appear to of the different subclasses should be
suppress differentiation of B cells measured if the total IgG level is normal but
into plasma cells. the clinical picture suggests
o Secondly, T cells may fail to provide immunoglobulin deficiency.
adequate help to support terminal  Most IgG antibodies directed against
differentiation of B cells. protein antigens are of the IgG1 and IgG3
o Finally, there appears to be a sub-classes, whereas most IgG antibodies
primary defect in the B-cell line in against carbohydrate antigens are IgG2 or
some patients. IgG4.
 CVI is often a diagnosis of exclusion, where  Thus, deficiencies involving IgG1 or IgG3
an immunodeficiency is present with no lead to a reduced capability of responding
specific genetic defect defined. to protein antigens such as toxins, whereas
 CVI can usually be effectively treated with selective deficiencies of IgG2 can result in
intramuscular or intravenous impaired responses to polysaccharide
immunoglobulin preparations. antigens, which cause recurrent infections
 However, because of their low levels of with polysaccharide-encapsulated bacteria
secretory IgA, patients are still susceptible such as Streptococcus pneumoniae and H
to respiratory and gastrointestinal influenzae.
infections; the clinician should be vigilant  A variety of genetic defects have been
for these infections and treat them associated with IgG subclass deficiency.
vigorously with antibiotics. These include heavy-chain gene deletions
and transcriptional defects.
 The most common subclass deficiency is
IgG4, with IgG1 deficiency being the least
common, although IgG4 subclass
deficiency may have the least clinical
significance.
Category 1: Combined
Immunodeficiencies
This category contains diseases in which there are

Isolated IgG Subclass Deficiency defects in both humoral (B cell) and cell-mediated
IgG subclass deficiencies are conditions where the (T cell) immunity.
level(s) of one or more of the four IgG subclasses is  These deficiencies result from mutations
(are) more than two SDs below the mean age- that affect development of both types of
appropriate level. lymphocytes or cause defective interaction
 Normally, about 70% of the total IgG is between the two antigen-specific limbs of
o IgG1, 20% the adaptive immune system.
o IgG2, 6%  Because helper T-cell functions are
o IgG3, and necessary for normal differentiation and
o 4% IgG4. antibody secretion by B cells,
 Therefore, a deficiency of a single subclass  a severe defect of T-cell function will have
may not result in a total IgG level below the
[BSMT 2B- 2020-2021- Group 4] Page 10

effects on immunoglobulin levels as well. adapter molecules, downstream

Combined deficiencies are referred to using a messengers or transcription factors
shorthand notation of T+/–B+/–NK+/– with involved in TCR signaling.
the + or – superscript denoting whether or not  Depending on the underlying genetic
each cell type is present in the deficiency. defect an individual with SCID may
have:
Severe Combined Immunodeficiency o Total loss of b-cell and t-cell
(SCID) o lost only t-cell , but there is b-cell
The most serious of the congenital present
immunodeficiency's  TH Dependent B cell activation
It is a inherited format of immunodeficiency T cell helps the B cell to get
where T-cell and B-cell mediated immune activated and the activation
response is lacking. these signal will tell to perform
R The disease resulting absence of T-cell or somatic or undergo class
significantly impaired T-cell function. switching.
R This defect combined with some disruption  TH Independent B Cell
of antibody responses. Will not happen when there is
It results from genetic defects that lead to a independent B cell activation
virtual or absolute lack of T-cell function “T cell mediated and B cell activation
cells in the periphery leads to class switching”
These defects target steps that occur early  Infants born with SCID experience
in severe recurrent infections that without
T- cell development or that affect the an early, aggressive treatment , can
quickly prove fatal.
Stem cells that feed the lymphoid lineage  Child having SCID may suffer:
Defective cytokine signaling in T-cell Oral candidal yeast infection,
precursor caused by certain cytokines, pneumonia, and diarrhea these
cytokine receptor or regulatory molecules are the most common
that control their expression. manifestation.
Premature death of lymphoid lineage They have to be kept in isolated
Defective VDJ rearrangement in developing area which is germ free
lymphocytes, caused by mutations in the  The immune system is compromised in
genes for RAG1 and RAG2 , or other SCID patients that common microbes
proteins involve in the rearrangement and even live attenuated vaccines can
process. cause persistent infection and life-
RAG1 AND RAG2 are crucial for VDJ threatening disease.
recombination The treatment option for SCID is
RAG 1 and RAG2 mutated for VDJ Bone marrow transplantation.
recombination does not happen and T-cell Take note: Patients with SCID
they lack diversity that caused SCID die before they are 2 years old.
 IL2 receptor gamma gene located in X-
DISRUPTIONS IN PRE TCR OR TCR SIGNALING Chromosome is the most common form
DURING DEVELOPMENT of disease. (It is a X-Linked SCID)
Caused by mutations in tyrosine kinase,  The IL2 RG gene codes for a protein
[BSMT 2B- 2020-2021- Group 4] Page 11

chain called the common gamma chain  Diseases in this category are typically
that is common to receptors for caused by defects in cell-mediated
interleukins-2, 4, 7, 9, 15 and 21. immunity,
IL7- B-cell development  which indirectly lead to problems with the
IL15- NK cell development other branches of the immune response.
 AID is important cause in SCID  Often, these diseases can result from
It is important for somatic abnormalities at different stages of T-cell
hypermutation and class development.
switching  Many different molecular defects can result
 ADA in a similar clinical picture (as in SCID). This is
due to lack enzyme adenosine because T cells provide helper functions that
deaminase are necessary for normal B-cell development
PURINE-NUCLEOSIDE PHOSPHORYLASE and differentiation
(PNP) DEFICIENCY
 Is a disorder of the immune system
(primary immunodeficiency)
 PNP deficiency is a rare autosomal
recessive trait
 The condition present in infancy
are:
o Chronic pulmonary
infections
o Oral or cutaneous
candidiasis
 diarrhea
 skin infection
urinary tract infections,
The numbers of T-cells progressively
decreases
2/3 of PNP- deficient patients also have
neurological disorder
It can be confused with neonatal HIV
infection
NOTE: Hematopoietic transplantation (HSCT)
is the only treatment option for the severe
immune deficiency.
Category 2: Combined
Immunodeficiencies With
Associated or Syndromic Features
This category differs from Category 1 in that

Wiskott- Aldrich Syndrome (WAS)
the diseases in Category 2 are characterized by
nonimmunologic features in addition to the
combined immunodeficiency.
[BSMT 2B- 2020-2021- Group 4] Page 12

defect, and located on the X chromosome,

region p11.
 In controlling the thrombocytopenia,
SPLENECTOMY can be very valuable.
TREATMENT:
 splantation of bone marrow
 Cord blood stem cells from HLA identical
sibling
Laboratory Features:
 Decrease in platelet number and size with
prolonged bleeding time.
 The bone marrow contains normal or
somewhat increased number of
megakaryocytes.
 Abnormalities in both cellular and humoral
branches of the immune system related to a
6. Rare X-linked recessive syndrome that is general defect in antigen processing
defined by the triad of immunodeficiency,
eczema, and thrombocytopenia. DIGEORGE ANOMALY
7. Usually lethal in childhood because of :  DiGeorge Anomaly is a developmental
Infection abnormality of the third and fourth pharyngeal
Hemorrhage pouches that affects thymus development in
Malignancy the embryo.
8. Also been described such as X-linked  All organs derived from these embryonic
form of thrombocytopenia structures can be affected.
 Associated abnormalities includes;
9. Patients display severe deficiency of the
naturally occurring IgM antibodies to ABO Mental Retardation
blood group antigen Absence of Ossification of the hyoid bone
(ISOHEMAGGLUTININS). Cardiac Anomalies
10. Absence of isohemagglutinins is the Abnormal Facial Development
most consistent laboratory finding in WAS Thymic Hypoplasia
and is often used diagnostically.  Many patients with a partial Digeorge Anomaly
11. Patients also have persistently have only a minimal thymic defect, and thus,
increased in levels of serum alpha- near normal immune Function.
fetoprotein, which can also be a useful  However, about 20% of Children with a defect
diagnostic feature. of the Third and Fourth pharyngeal pouches
12. Primary molecular defect in the have a severe and persistent decrease in T-cell
numbers.
syndrome appears to be an abnormality
to the integral membrane protein CD43,  Severely affected children usually present in
which is involved in the regulation of the neonatal period with tetany ( caused by
protein glycosylation. hypocalcemia resulting from
13. _____________, the gene responsible
for
[BSMT 2B- 2020-2021- Group 4] Page 13

hypoparathyroidism) or manifectations of autosomal recessive syndrome

cardiac defects. characterized by Cerebellar Ataxia
(involuntary muscle movements) and
 The immunodeficiency associated with the Telangiectasias (capillary swelling resulting
DiGeorge anomaly is a quantitative defect in in red blotches on the skin), especially on
Thymocytes the earlobes and conjunctiva.
 The immunodeficiency of DiGeorge Syndrome  Abnormal genes produce a combined
can be treated with fetal thymus defect of both humoral and cellular
transplantation. immunity.
 Bone Marrow Transplantation has also been  Antibody response to antigens, especially
successful in some patients, as has polysaccharides, is blunted, although the
administration of thymic hormones. pattern can be quite variable.
 Death usually occurs in early adult life from
either pulmonary disease or malignance.
 Apparently essential to the recombination

process for genes in the immunoglobulin
superfamily.
 The AT gene is located on Chromosome 11,
region q22.
 Most Patients with DiGeorge Syndrome  Abnormal results in a defective kinase
show a deletion in Chromosome 22, involved ;
region q11, although this anomaly is not  DNA repair and in cell cycle control
required for diagnosis.  Patients lymphocytes often exhibit
 The q11 region of Chromosome 22 deletion chromosomal breaks and other
is also associated with velocardiofacial abnormalities involving the TCR genes in T
syndrome (VFS) and other syndromes. cells and immunoglobulin genes in B cells.
Ataxia- Telangiectasia  The Syndrome is associated with an even
greater risk of lymphoid malignancy than
other immunodeficiency syndromes,
presumably because the failure to properly
repair DNA damage leads to the
accumulation of mutations.
➢ Ataxia-Telangiectasia (At) is a rare Effective therapy:
[BSMT 2B- 2020-2021- Group 4] Page 14

 Allogeneic bone Marrow transplantation. Immunodeficiency with hypopigmentation (

CATEGORY 4: DISEASE OF IMMUNE loss of skin color)
DYSREGULATION Caused;
Mutation in the LYST gene
Reduced number of natural killer (NK) cells and
Neutrophils.
Increased production of Inflammatory proteins.
Peripheral Blood Smear from patients with
Chediak Higashi Syndrome shows
_______ inclusion attributed to Enlarge
lysosomes.
 Normal numbers of T or B cells but with

reduced control over their functions.
 Also have features of Autoimmunity
The Autoimmune Lymphoproliferative

Syndrome ( ALPS)
 May involved in apoptosis. Defective apoptosis

in the thymus may lead to autoreactive cells in
the circulations.
 The CD25 deficiency is manifested by a lack of
T regulatory (TREG) cells, which leads to Category 5: Congenital Defects of
_________ and __________. Phagocyte Number, Function, or Both
 Mutation; Clinical presentation.
Classifies;
CHEDIAK HIGASHI SYNDROME
PIDS, which are characterized by
abnormalities in Phagocytic cells.
_______ Play a crucial role in the

immediate and nonspecific response to
invading organisms by responding before
specific antibody and cell-mediated
immune response can be mounted.
[BSMT 2B- 2020-2021- Group 4] Page 15

 Neutrophils are even more effective at necessary for normal bacterial killing.
ingesting and killing organisms coated with
specific antibody and thus continue to play  Three different autosomal recessive genes
are involved and all affect subunits of
a important role in host defense even after
nicotinamide adenine dinucleotide
an adaptive immune response is
phosphate (NADPH).
established.
 Neutrophils must adhere to vascular

endothelial lining cells, migrate through the
capillary wall to a site of infection, and
ingest and kill the microbes.
 Defect affecting each of these steps can

lead to an increased susceptibility to
pyogenic infections.
Chronic Granulomatous Disease

(CDG) A genetic defect in any of the several
components of the NADPH oxidase system
can result in the CDG phenotype by making
the neutrophil incapable of generating an
oxidative burst.
It was historically diagnosed by measuring

the ability of a patient’s neutrophils to
reduce the dye nitroblue tetrazolium
(NBT).
SYMPTOMS:
Recurrent suppurative infections

Pneumonia
 Is a group of disorders involving
Osteomyelitis
inheritance of either an X-linked or
Draining adenopathy
autosomal recessive gene that affects
Liver abscesses
neutrophil microbiocidal function.
Dermatitis
 Most common and best characterized of Hypergammaglobulinemia
the neutrophil abnormalities. CATALASE-POSITIVE ORGANISMS INVOLVED:
Staphylococcus aureus
 Results inability of the patient’s neutrophils Burkholderia cepacia
to produce the reactive forms of oxygen Chromobacterium violaceum
[BSMT 2B- 2020-2021- Group 4] Page 16

FUNGI INVOLVED:  Motility

Aspergillus  Aggregation
Nocardia  Chemotaxis
TREATMENT:
 Therapy with with granulocyte Endocytosis by the affected leukocytes
 Continuous use of antibiotics
 Bone marrow transplantation or use of
peripheral blood stem cells
Other Microbiocidal Defects
Several other defects can result in impaired
neutrophil.
_______________ deficiency that can lead
to an inability to generate enough NADPH SYMPTOMS:
to supply reducing equivalents to the Delayed wound healing
NADPH oxidase system. Chronic skin infection
Intestinal and respiratory tract infections
Shortfall leads to a defect in
hydrogen peroxide production and a periodontitis
clinical picture similar to that CDG. DIAGNOSIS:
Myeloperoxidase Deficiency is relatively Defect in CD18 can be diagnosed by
common, occurring in about 1 in 3,000 detecting a decreased amount of CD11/18
person in United States. antigen on patient leukocytes by FLOW
Deficient patients may have recurrent CYTOMETRY.
candidal infections. OTHER TYPE:
Defect of neutrophil secondary granules
have been described also. The molecular LAD II - in this disorder a carbohydrate
nature of the defect is unknown. molecule involved in adhesive interaction,
Leukocyte Adhesion Deficiency CD15s, or sialyl-Lewis X, is deficient.
(LAD)
Category 6: Defects in Innate Immunity
Deficiency in protein CD18, which is a
component of adhesion receptors on
neutrophils and monocytes (CD11b or  New part of the PID classification, Mirroring
CD11c) and on T cells (CD11a). the explosive increase in knowledge and
understanding of the innate immune
The CD18 deficiency is transmitted system.
through autosomal recessive
inheritance and has a variable  At least one disease, Chronic
expression. mucocutaneous candidiasis.
The defect may lead to:  As a T-cell defect
Abnormal adhesion
[BSMT 2B- 2020-2021- Group 4] Page 17

cytokines IL-1 and IL-18.

Genetic Defects includes;
 The Hyper IgD syndrome also referred to as
periodic fever syndrome and Muckle-
Wells syndrome.
 Hyper Igd is caused by; A deficiency of
Mevalonate kinase
DIAGNOSIS
 Researchers identified two forms of this  Clinical presentation of Recurrent Fevers,
entity involving mutations in genes coding  IgD Testing.
for IL-17.
Caused by;
 Other rare entities classified under this
 A mutation in the CIAS1 gene coding for
heading include; Mutations in Toll-like Cryopyrin,
receptors (TLRs).
a component of the Inflammasome.
Example; TLR signaling pathways, such
 Patients may present with Urticaria and
as IRAK4 deficiency, can also occur.
Amyloidosis.
Both types of defects can lead to
bacterial infections.
Defect not Involving the Inflammasome include;
 Diagnosing these entities is based upon  Tumor Necrosis Factor (TNF)
clinical presentation, which leads to
 receptors-associated periodic syndrome (
molecular analyses to identify specific TRAPS) and
genetic mutations.
 Early-Onset Inflammatory bowel disease (
Category 7: Autoinflammatory Disorders
IBD).
TRAPS
 Autoinflammatory disorders are subdivided  Caused by a mutation in the TNFRSF1A
into two classifications; gene, which codes for a TNF receptors
 Those involving the inflammasome and and may result in recurrent fevers, as well as
 noninflammasome conditions ocular and joint Inflammation.
Inflammasome IBD
 A protein Oligomer that contains caspase  Early Onset IBD is Caused by Mutations in
enzymes and other proteins associated genes coding for IL-10 or its receptors.
with apoptosis Muckle-Wells syndrome and TRAPS
 Location:  Shows autosomal dominant inheritance,
Primarily in myeloid cells and may be whereas Hyper IgD
activated by various microbial substances. and early-Onset IBD are Autosomal recessive.
Once activated;
 The inflammasome stimulates the
production of the proinflammatory
[BSMT 2B- 2020-2021- Group 4] Page 18

❖ Deficiencies of later component of

clompent (C5-C9) are often associated with
recurrent Neisseria meningitidis
Category 8: Complement Deficiencies
 Complement consist series of proteins that

work in cascade to assist in antibody  C1 deficiency esterase inhibitor has been
destruction of cell. found in the patients with hereditary
neuroangioedema.
 It is also part of innate immune system and
works as part of the inflammatory system  Most complement deficiencies appear to
directly eliminate a potential pathogen. be inherited in an autosomal recessive
manner and are likely caused by random
 Deficiencies in the earl complement
changes in DNA nucleotide sequence as
components; C1q, C4, and C2 are usually
associated with lupus-like syndrome. opposed to genetic detection.
Category 9: Phenocopies of Primary
Immunodeficiencies
 This category comprises a new classification

of PIDS.
 Disorders that fall into this Category have;
Inherited genetic component but also include
an acquired component, such as;
(Lupus syndrome)  Somatic mutations or autoantibody
 C2 deficiency - believed to be the most production.
common complement component This disease is included by a genetic mutation
syndrome. in the AIRE gene, but also involves an antibody
 C3 deficiency - also have lupus-like clinical to either ( or both) IL-17 and IL-22
presentation, but is more likely to involve Mutations in the nRAS or kRAS genes are also
recurrent infections with encapsulated associated with diseases that fall into this
organisms. category.
[BSMT 2B- 2020-2021- Group 4] Page 19

[BSMT 2B- 2020-2021- Group 4] Page 20

Laboratory Evaluation of Immune polysaccharide antigens, such as those in

Dysfunction the H influenzae and S pneumoniae
vaccines.
Screening test
Delayed hypersensitivity-type skin reactions
used for the initial evaluation of a
suspected immunodeficiency state can be used to screen for defects in cell-
performed routinely in any hospital mediated immunity
laboratory generally performed by the clinician and
not by laboratory personnel
Measurement of the levels of serum IgG,
IgM, and IgA and levels of the subclasses the prototype is the tuberculin skin test
of IgG are used to screen for defects in An antigen to which most of the population
antibody production. has been exposed, such as candida,
By the age of 2, a child should have mumps, or tetanus toxoid, is injected
naturally occurring IgM antibodies intradermally
against ABO blood group antigens. Which The presence of induration 48 to 72 hours
means the absence of these antibodies later indicates a cell-mediated immune
suggests an abnormal IgM response response
A negative test is not always informative
An overall assessment of antibody- because the patient may not have been
mediated immunity can be made by previously exposed to the test antigen
measuring antibody responses to antigens
to which the population is exposed Screening for complement deficiencies
normally or following vaccination
usually begins with a CH50 assay.
measuring the titer of the specific antibody
This procedure determines the level of
produced in response to immunization with
functional complement in an individual
a commercial vaccine such as
diphtheria/tetanus. Undetectable CH50 levels may indicate
a deficiency of a specific component
o Unimmunized child: 2 weeks
development of tetanus or complement consumption and do not,
by themselves, indicate a complement
diphtheria antibodies
deficiency
o Immunized patient: 4 to 6 weeks
response to a booster injection Defects in neutrophil oxidative burst activity
wide range of other protein and
polysaccharide antigens can also be used may be detected by a flow cytometric
in these tests assay
evaluate a possible IgG subclass Neutrophils labeled with DHR are
deficiency. stimulated to undergo an oxidative
IgG1 and IgG3 isotypes normally burst by exposure to a mitogen
respond to protein antigens, such as The oxidative burst will reduce the
tetanus and diphtheria DHR, producing fluorescence, which
While IgG2 normally responds to can be measured objectively by flow
[BSMT 2B- 2020-2021- Group 4] Page 21

cytometry Flow cytometry

Flow cytometry can also be used to
confirm a diagnosis of LAD type 1 by Enumeration of classes of lymphocytes in
looking for the expression of the CD18 the peripheral blood is performed by flow
antigen. cytometry
Before flow cytometric analysis, antibodies
Confirmatory test
to antigens specific for different types of
lymphocytes are labeled with a fluorescent
In confirmatory test, specialized testing is
probe.
required
These antigens are generally referred to by
a cluster of differentiation (CD) number
Lymphocytes can then be assigned to
specific types based on antigen expression:
B cells (CD19), T cells (CD3), T helper (Th)
cells (CD3/CD4), cytotoxic T cells
(CD3/CD8), and NK cells (CD16 or CD56).
Absence or profound decrease in the
number of CD3 cells = DiGeorge
syndrome
Absence of CD19+ B cells =

Btk deficiencies
Secondary deficiencies
Absent B cells = patients treated with

rituximab, a monoclonal anti CD20
antibody
Note: no CD19+ B cells
are detectable
Most of the genes associated with PIDs
have been identified and localized
Genetic testing of family members of
affected patients may be helpful in
determining who may be at risk of
developing the disease or passing it on to
offspring
T-cell function
Phytohemmagglutinin (PHA) or
Concanavalin A (Con A)
A mitogen is a substance that stimulates
[BSMT 2B- 2020-2021- Group 4] Page 22

mitosis in all T cells or B cells, regardless of related defects leading to SCID - Severe
antigen specificity Combined Immune Deficiency
T cell response may be measured by  DiGeorge and other non-SCID diseases,
quantitating the uptake of radioactive such as Omenn syndrome, have also been
thymidine, a precursor of DNA, increased detected using this method
thymidine uptake suggest cell division
and activation.
antigen- og mitogen- stimulated T-cell Evaluation of Immunoglobulins
activation has been measured without the the basic immunoglobulin unit consists of two
use of radioactive materials identical :
heavy chains and
3 assays for diagnostic use: Quantiferon TB assay, two identical light chains,
T-Spot assay, and Cylex ImmuKnow assay covalently linked by disulfide bonds.
The structure of the heavy chain :
Quantiferon TB assay and T-Spot assay defines the class, or isotype, of the antibody (e.g.,
- measure an individual’s response to heavy chain in IgG, heavy chain in IgM, etc.).
Mycobacterium tuberculosis antigens The two types of light chains
can each occur in combination with any of the
Either of these assays may be used as an heavy-chain types.
in vitro assessment of exposure to M -The heavy and light chains each contain constant
tuberculosis and variable regions.
The initial tests used to screen for the presence
Cylex ImmuKnow assay - measures total T-cell of a monoclonal gammopathy are :
activity serum immunoglobulin levels and SPE
-Quantitative measurement of immunoglobulin
This test uses the mitogen PHA to activate levels in the serum is routinely performed by
T cells
nephelometric methods, or in smaller laboratories,
This test is a general measurement of T-cell by radial immunodiffusion (RID)
function and is often used to monitor Serum Protein Electrophoresis (SPE)
individuals receiving immunosuppressive is a technique in which serum proteins are
therapy separated on the basis of their size and electric
charge
Newborn Screening for Immunodeficiencies SPE results in five regions:
 TCR excision circles (TRECs) albumin, as well as the alpha 1, alpha 2, beta, and
gamma globulins. IgG, IgM, IgD, and IgE migrate in
 TRECs, identified by quantitative PCR are the gamma globulin region, whereas IgA migrates
present in T cells that have undergone as a broad band in the beta and gamma regions.
alpha-beta receptor gene rearrangements Bone Marrow Biopsy
 They are the genetic material that has been -indicated in any evaluation of a monoclonal
removed from the germline DNA during gammopathy or immunodeficiency state
alpha VJ and beta VDJ recombination
-A bone marrow biopsy can take about 60 minutes.
 Their absence indicates a lack of functional
Bone marrow is the spongy tissue inside your
T cells, allowing early identification of T-cell
bones.
[BSMT 2B- 2020-2021- Group 4] Page 23

-There are two types of marrow: RED and

YELLOW
-Red marrow is mainly found in your flat bones
such as your hip and vertebrae. This type of bone
marrow contains hematopoietic stem cells, which
are the stem cells that form blood cells.
-Yellow bone marrow is involved in the storage of
fats. The fats in yellow bone marrow are stored in
cells called adipocytes.
Family History
-The importance of obtaining a full family history
as part of the PID diagnosis process cannot be
overemphasized. The mode of inheritance, if
know, can rule in or rule out many of the PIDs.
-The disease may be X-linked, such as in the
common gamma chain mutation, WAS, or the
CD154 deficiency; autosomal dominant, such as in
Hyper IgE or Muckle-Wells syndrome or autosomal
recessive
-The family is required for a final diagnosis.
[BSMT 2B- 2020-2021- Group 4] Page 24

IMMUNOLOGY AND
AUTOIMMUNITY SEROLOGY (IS)
AUTOIMMUNTIY / GROUP 3 / BSMT 2B
AUTOIMMUNITY
 The absence of another well-defined cause of the
History: disease.
Paul Ehrlich
- dictum: horror autotoxins or fear of self- Mechanism involved in the induction of self-tolerance
poisoning- (self-tolerance)
Central tolerance
- the body has an aversion to producing an
immune response to its own antigens (self induced by clonal deletion
-antigens) positive and negative selection processes
Autoimmunity eliminate cells with the potential to react with
self-antigens.
- mechanism by which the body (immune
develops in the thymus and bone marrow
system) distinguish self from non-self
during fetal development.
- condition in which structural or functional
Peripheral tolerance
damage is produced by action of
immunologically competent cells or antibodies induce by clonal anergy
against the normal components of the body occurs in the circulation and mature
Autoimmune disorder lymphocytes
it is mediated by Treg and suppressor T-cells
- used when demonstrable immunoglobulins
(autoantibodies) or cytotoxic t-cells display Triggers for Autoimmune disorders:
specificity for self- antigens (autoantigens) and
contribute to the pathogenesis of the disorder. Molecular mimicry
Autoimmune diseases - an antigen found on another organism that cross
- leading cause of chronic illness and death reacts with self-antigens
EXAMPLES:
AUTOIMMUNE INFECTIOUS
DISEASE AGENT
Serge Metalnikoff (1900) reported that some animals Acute rheumatic Streptococcus pyrogens
were able to form antibodies against their own fever
spermatozoa. Type 1 DM CMV, Hepa C virus,
measles
Paul Ehrlich (1901) rejected the concept that an Reactive arthritis Klebsiella (HLA-B27)
organism's immune system could attack the organism's Myasthenia Gravis Poliovirus
own tissue calling it "horror autotoxicus“ Hidden antigens
Julius Donath and Karl Landsteiner (1904)reported also known as sequestered, occult, cryptic
autoantibodies can cause disease by showing that antigens
autoantibodies (‘hemolysins’) caused paroxysmal cold EXAMPLES: -Sperm cells
hemoglobinuria. cornea: immuno-privilege site
brain
Ernst Witebsky and Noel Rose (1956) were able to uterine environment
induce an experimental autoimmune thyroiditis mediated
by autoantibodies. they do not normally circulate in the blood and
hence do not have contact with the mononuclear
3 requirements to call it Autoimmunity phagocyte system
they spread by epitope spreading
 The presence of an immune reaction

Altered antigens
specific for some self antigen or self tissue.
 Evidence that such a reaction is not also known as neoantigens (modified self
secondary to tissue damage but is of -antigens)
primary pathogenic significance. caused by chemical, physical, or biological
processes
IMMUNOLOGY AND
AUTOIMMUNTIY / GROUP 3 / BSMT 2B responsive to self-antigens
Loss of immunoregulatory function by T
not true antigens lymphocytes subset
T reg, CD44, CD25+ T cells decreases activity
Mutation of immunocompetent cells
of activated t cells
seen in aging
Immunopathologic mechanism:
 In Self-tolerance, our immune system
Immune complex recognizes self-produced antigens as a
- post streptococcal glomerulonephritis non-threat while appropriately mounting
Cytotoxic T cells a response to foreign substances. This
ADCC (antibody-dependent cellular cytotoxicity) balance of immunological
defense and self-tolerance is
Immunological symptoms of Autoimmune Diseases: critical to normal physiological
function and overall health. So,
T- lymphocytes which demonstrate reactivity failure of the immune system to
against autoantigen "tolerate" self- tissues or loss of
Presence of autoantibody self- tolerance can result in
Hypergammaglobulinemia pathological autoimmune states
Generalized hypocomplementemia leading to debilitating illness and
sometimes death.
Elevated chemotaxis
 Self-toleranceis the lack of
Factors influencing development of autoimmunity an immune response, particularly
by T and B lymphocytes, to
Genetics antigens that are normal
Gender Age constituents of the
Environmental exposure Race
.
Presence of chronic infectious diseases
*In order for self-tolerance to develop, lymphocytes
ETIOLOGY OF AUTOIMMUNE DISEASE must be “educated” so they can distinguish between self-
antigens and foreign antigens. This education takes place
Under normal circumstances, our immune system is able to at two levels: central and peripheral.
differentiate between “self” and “nonself” or “foreign,” so A. Two levels of educating lymphocytes:
that self-antigens are not destroyed.
 Central tolerance
occurs in the central or primary
lymphoid organs. (the thymus for t cells,
and the bone marrow for b cells)
In here, note that this is where the negative
and positive selection of thymocytes occurs.
It is a part of the T cell development which is
the Double-Positive Stage. Recall that when
we say double positive stage, our thymocytes
already express both CD4 and CD8 antigens.
T cells bearing the CD4 receptor are mainly
T helper (Th) cells, whereas the CD8+
population consists of cytotoxic T cells.
1. SELF TOLERANCE THYMUS
 the ability of the immune system to accept self-
antigens and not initiate a response against them. =In a process called negative selection,
(Autoimmune disease is thought to result from a T cells that express T-cell receptors
loss of self-tolerance) (TCRs) with a strong affinity for these
self antigens are deleted by apoptosis.
 a type of immunologic tolerance, or a state of  a positive selection process
immune unresponsiveness that is directed against a takes place that allows only DP cells with
specific antigen, in this case, a self-antigen.
functional TCR receptors to survive.
IMMUNOLOGY AND
epithelial cells. If very strong bonding
occurs, cells are eliminated by
T cells must recognize foreign antigen in
apoptosis. If very weak or no bonding
association with class I or class II MHC
occurs, cells are also eliminated. It
molecules. Positive selection of thymocytes
should be intermediate binding only.
in the cortex. Double-positive (CD4+ and
*During this process, some of the self-
CD8+) thymocytes interact with thymic
reactive CD4+ T cells are not deleted,
but instead differentiate into T regulatory
(Treg) cells that can specifically inhibit
apoptosis. Self-reactive B cells in the
immune responses to self-antigens.
periphery can be deleted by apoptosis, be
BONE MARROW rendered anergic after repeated stimulation
with self-antigens, or receive inhibitory
As B cells mature in the bone marrow, those signals through receptors such as CD22.
with receptors having a strong affinity for Its main purpose is to ensure that
self-antigens are eliminated by apoptosis. self-reactive T and B cells which
Some self-reactive B cells are not deleted: escaped
rather, they are stimulated to rearrange their central tolerance do not
immunoglobulin genes so that their B-cell cause autoimmune disease.
receptors are no longer antigen specific. This
In some individuals, self-tolerance can fail even
process is known as receptor editing.
after this second layer of protection; if this
happens, autoimmunity can arise. The
Anergy= B cells that possess receptors that only development of autoimmune disease is thought to
weakly recognize self-antigens are induced to be caused by complex interactions between
downregulate the expression of their receptors and genetics, exposure to environmental factors, and
develop a specific state of unresponsiveness to the defects in immune regulation.
antigens The major factors that are believed to
*This process is not totally effective, however, and some contribute to autoimmunity:
self-reactive lymphocytes manage to escape to the
Genetics
secondary lymphoid organs such as the lymph nodes and
Autoimmune diseases are often more prevalent
spleen. Therefore, a second level of protection is needed. among family members than among unrelated
 Peripheral tolerance= lymphocytes that individuals and are more prevalent among
recognize self-antigens in the secondary monozygotic (genetically identical) twins than
lymphoid organs are rendered incapable of dizygotic (non-identical) twins or siblings
reacting with those antigens. Peripheral The strongest link found is between the HLAB27
tolerance of T cells can result from anergy allele and the development of
caused by the absence of a costimulatory ankylosing spondylitis, an autoinflammatory
signal from an antigen-presenting cell (APC) disease that affects the spine. Individuals who
or binding of inhibitory receptors such as possess HLA-B27 have about a 100 times greater
CTLA-4 (a molecule that prevents T-cell chance of developing the disease than individuals
activation). Peripheral T-cell tolerance can who do not have that allele.
also result from inhibition by Tregs or death
by Differences in MHC genes are thought to
influence the development of autoimmune
disease. (because the specific structure of the
MHC molecule can determine whether or not a
self-antigen can attach to the peptide-binding
cleft of the molecule and subsequently be
processed and presented to T cells)
Class II MHC molecules can sometimes be
abnormally expressed on cells where they are not
typically found. (which results now in the
presentation of self-antigens for which no
tolerance has been established)
Polymorphisms in some non-MHC genes can
also be associated with development of
autoimmune disease. Many of these
IMMUNOLOGY AND
AUTOIMMUNTIY / GROUP 3 / BSMT 2B PTPN22 gene- which has a role in T-
and B-cell receptor signaling.
genes influence the development and regulation of IL2RA gene- involved in T-cell
immune responses. activation and maintenance of Tregs.
EXAMPLES INCLUDE: CTLA4 gene- which has an inhibitory
effect on T-cell activation.
BLK gene - which is involved in B-cell activation
and development.
AIRE (autoimmune regulator) gene-which tend to develop autoimmunity at an earlier age
promotes the development of T-cell tolerance in and have a higher risk for acquiring more than
the thymus. one autoimmune disease as compared with men.
 Although most autoimmune diseases involve have been found to have higher absolute CD4+
multiple genes (~20 to 30), single-gene mutations T-cell counts and higher levels of circulating
that can be inherited in a Mendelian fashion have antibodies than men.
been associated with rare autoimmune disorders.
*These observations suggest that there is a
REMEMBER: Inheritance of specific genes may make an hormonal influence on the development of
individual more susceptible to a particular autoimmune autoimmunity. Studies on the effects of
disease, but genetic makeup is not totally responsible hormones have shown that estrogens ( a female
because the majority of people with a particular gene will hormone) tend to direct the immune system in
not develop autoimmunity. favor of a type 2 helper cell (Th2) response, So
there would be more B-cell activation and
Which is evident in these ones…..
antibody production whereas androgens favor a
 The concordance rate among monozygotic twins type 1 helper cell (Th1) response with activation
(i.e., the presence of autoimmune disease in both of CD8+ T cells).
members of a pair of identical twins) is only
Example: Synovial fluid levels (SF) of
between 20% and 30% for most autoimmune
proinflammatory estrogens relative to androgens
disease.
are significantly elevated in both male and
female rheumatoid arthritis (RA) patients.
 The prevalence and severity of many autoimmune
diseases vary in different geographic locations. *estrogens as enhancers at least of the
humoral immunity and androgens and
progesterone (and glucocorticoids) as
natural immunosuppressors.
 Prolactin-a hormone that stimulates production
of breast milk in pregnant and nursing women,
can stimulate both humoral and cell-mediated
immune responses.
2. Tissue Trauma and Release of
Cryptic Antigens
*When immunologic tolerance to self-
antigens occurs during the early
development of lymphocytes in the
This variance suggests that environmental and thymus and bone marrow:
other factors also play a role in the development
of autoimmune disease.  Some Self-antigens may be cryptic, or hidden
within the tissues of the host.
 Other Endogenous and Environmental Factors
 T and B lymphocytes are shielded from these
1. Hormonal Influence sequestered antigens and are not educated to
WOMEN become tolerant to them.
 2.7 times more likely to acquire an autoimmune  At a later time in life, inflammation or tissue
disease than men; in fact, about 78% of patients trauma could cause the cryptic antigens to be
with autoimmune diseases are of female gender. released and to suddenly be accessible to the
uneducated lymphocytes, triggering an immune
response.
IMMUNOLOGY AND
AUTOIMMUNTIY / GROUP 3 / BSMT 2B to sperm after a vasectomy, and autoantibodies to
DNA following damage to skin cells by
overexposure to UV rays from the sun.
 This concept has also been referred to as
immunologic ignorance and may be responsible *Cornea-(an immuno-privileged organ)
for the production of autoantibodies to the lens of
the eye following an ocular injury, autoantibodies
3. Microbial Infections
 Bacteria, viruses, and other infectious pathogens

may be able to trigger autoimmune responses in a  This expansion of the immune response to
variety of ways. unrelated antigens has also been termed “epitope
spreading.”
 A principal means by which microbes are thought
to accomplish this is through molecular mimicry.  SUPERANTIGENS- proteins that are produced
by various microbes that have the ability to bind
1. MECHANISMS OF HOW to both class II MHC molecules and TCRs,
MICROBES TRIGGER AUTOIMMUNITY: regardless of their antigen specificity.
MOLECULAR MIMICRY - refers to the fact that
many bacterial or viral agents contain antigens  Examples:
that closely resemble the structure or amino acid 1.staphylococcal enterotoxins that cause food
sequence of self-antigens. Exposure to such poisoning and toxic shock syndrome=these
foreign antigens may trigger immune responses superantigens can act as potent T-cell mitogens
that cross-react with similar self-antigens. by activating a large number of T cells with
Example of this mechanism: different antigen specificities. If some of these T
Association between the gram-positive cells possess specificity for a self-antigen,
bacterium Streptococcus pyogenes and autoimmune responses might result.
rheumatic fever 2.some viruses, including the Epstein-Barr virus
Some patients who have acquired scarlet fever or (EBV) and cytomegalovirus (CMV), can cause
pharyngitis as a result of infection with S polyclonal activation of B cells.
pyogenes will proceed to develop rheumatic fever 4. Epigenetics and Modification of Self-
if they are not treated adequately with antibiotic.
Antigens
A second way that microbes might trigger
Epigenetics refers to modifications in gene
autoimmunity is through: expression that are not caused by changes in the
BYSTANDER EFFECT- In this mechanism, the original DNA sequence.
microbial organism does not have to share -So, these alterations in our gene expression are
structurally similar antigens with the host.
stable and can be inherited. They are thought to
Instead, the microorganism can induce a local
be triggered by exposure to environmental toxins,
inflammatory response that recruits leukocytes
and stimulates APCs to release cytokines that ingestion of harmful foods or drugs, or the aging
non-specifically activate T cells. (Some of the T process. These factors can induce epigenetic
cells that are activated may have specificity for changes by increasing or decreasing methylation
self-antigens) of cytosine bases, modifying histones, and
causing abnormal regulation by microRNAs.
These modifications can result in changes in the
level at which genes are expressed by affecting
their ability to be transcribed into mRNA, which
is subsequently translated into proteins that will
influence the phenotype of an individual. Over-
or under expression of certain genes in the
immune system may result in homeostatic
imbalances and a breakdown of self-tolerance,
leading to autoimmunity.
Interactions Between Factors
Although the precise etiology of autoimmunity is

unknown, there is much
IMMUNOLOGY AND
AUTOIMMUNTIY / GROUP 3 / BSMT 2B not sufficient by themselves to cause
autoimmune disease
evidence that suggests that this heterogeneous  FACTORS:
disease entity is caused by complex interactions
between genetic and environmental factor. Genes- Certain genes are thought to
make individuals more susceptible to
immune responses against self-antigens,
but are not sufficient by themselves to
cause autoimmune disease.
congenital complement component
Gender of the individual
tissue injury deficiency: hypocomplementemia
exposuretoinfectious Environmental factors:
microorganisms or other environmental exposure to UV light
agents certain medications
infectious agents
 As a result of this break in immunologic
Hormones (estrogen)
tolerance, autoreactive T cells recognize and
proliferate in response to self antigens and B cells Express itself as vasculitis involving many
develop into plasma cells that secrete organs
autoantibodies. This can result in the release of Immunologic mechanism for pathology
proinflammatory cytokines, which, when coupled type III hypersensitivity: immune complex
with dysfunctions in disease
immune-regulatory cells, perpetuate the depressed suppressor T cell function
autoimmune responses (and leads to)
autoimmune disease. Different forms of Lupus:
 Tissue injury in these disorders results from  Discoid (cutaneous) Lupus

hypersensitivity reactions that coin like lesions
appear when skin exposed to UV light
identified by skin biopsy
does not affect internal organs
10% of all lupus
 Systemic lupus
Affects the skin, joint, and almost any organ

or body system and more severe than discoid
Characterized by periods or “remission” and
“flare”
70% of all lupus
 Drug-induced lupus
involve autoantibodies to cell-surface receptors,  differs from the more chronic form of the
deposition of immune complexes that contain self- disease, in that symptoms usually disappear once
antigens, and cell-mediated cytotoxicity. the drug is discontinued.
 Most common in old people
 Most common drug implicated:
Procainamide
Hydralazine
B. SYSTEMIC AUTOIMMUNE DISEASE Chlorpromazine
Isoniazid
Quinidine
Systemic Lupus Erythematosus (SLE) Anticonvulsant such as: methyldopa,
phenytoin
Prototype of human autoimmune diseases Chronic oral contraceptives
systemic inflammatory disease marked by  Manifests as fever, arthritis, or rashes;
alternating exacerbations and rarely involves kidney
remissions.
Epidemiology: d) Neonatal Lupus
age of onset: 20-40 years old
gender: women > men (9:1)  Acquired from passage of maternal
race: African-Americans antibodies that can affect the skin, heart, and blood
Exhibits strong genetic tendency of the fetus/new born.
HLA association: HLA-DRW3, HLA-BB,  Characterized by rash; most common
HLA-DRW2  Possible complication: congenital heart
attack, hepatosplenomegaly, cytopenia
IMMUNOLOGY AND
- pericarditis, tachycardia, or ventricular
Criteria for Classification of SLE enlargement
Malar rash Discoid rash
Photosensitivity Oral ulcers
Nonerosive arthritis
Pleuritis or pericarditis Renal failure
Neurologic disorder Pleuritis with chest pain
Hematologic disorder
14. hemolytic anemia
15. leukopenia Laboratory:
-lymphopenia Screening test: Antinuclear Antibodies
- thrombocytopenia
Immunologic disorder Method of choice: fluorescent antinuclear
Positive anti-nuclear antibody antibody testing (FANA)
 detects wide range of antibodies and is positive
in about 95% of patients with lupus.
Note: if any four or more of the 11 criteria are present, Mostcommonlyused
a person shall be said to have SLE. substrates:
Clinical manifestations: - mouse kidney cell
Symptoms: -human epithelial cells (HEP-2)
fever  PRO: extremely sensitive test and
weight loss
malaise relatively easy to perform
weakness  CON: low diagnostic specificity o
anorexia
Antinuclear Antibodies (ANAs)
Joint involvement isotype: IgG>IgM or IgA

may cause positive DAT: direct anti-
most common manifestation
hands, wrist, knees globulin test -Responsible for leukopenia
manifest as: arthritis and
polyarthralgia and thrombocytopenia
symmetrical
-Responsible for biological false positive
Skin lesions
test for syphilis
second most common
red rash across nose and upper cheeks: examples:
butterfly rash Abs to DNA, DNA-histone, and
lupus: Latin for “red wolf” ENA
different from the rash of discoid lupus Abs to Nrnp
(central atrophy) Abs to PCNA
common trigger: exposure to UV light Renal Abs to SS-A and SS-B
involvement Abs to histones
renal failure: major cause of mortality Immunofluorescence Pattern in IIFA

deposition of immune complexes  Homogenous (solid, diffuse)
most severe form: diffuse proliferative - Whole nucleus fluoresces evenly green-gold
glomerulonephritis  Antibodies:
Anti-DNA
Cardiac involvement Anti-histone
IMMUNOLOGY AND
Anti-DNP
 Autoimmune diseases:  Peripheral (ring,
High titer: SLE membranous, shaggy,
Low titer: SLE, RA, Sjogren’s, thready)
Mixed connective tissue disease (MCTD)
 Sharp green-gold fluorescence of the
outer edge of nucleus with gradually
darkening inner border blending with a
dark nuclear center  Systemic sclerosis
 Antibodies:
Anti-DNA
Anti-lamin
 Autoimmune diseases:
SLE, Sjogren’s
 Speckled (mottled)
 Numerous rounds speckled of green-gold
nuclear fluorescence of various size
against dark background; “pepper dots”
 Antibodies:
Anti-ENA
 Anti-ribonucleoprotein
SLE, RA, SLE Autoantibodies:
Scleroderma,
 Anti-dsDNA
MCTD,
dermatomyosi Most specific for SLE but not diagnostic
ti s Its presence, along with low C3,
 Anti- smith (sm) is diagnostic for SLE
Highly IIFA fluorescence pattern:
specific Peripheral
marker for Homogenous
SLE Substrate: Crithidia luciliae
Nucleolar Anti- histone
Multiple rounds, smooth, green-gold
fluorescing nucleoli of various sizes Diagnostic for Drug-induced SLE
Antibodies: IIFA fluorescent pattern: Homogenous
Anti-nRNA Nonspecific:
 Scleroderma -70%SLE
 Sjogren -RA
 SLE -PBC
 Undiagnosed  Anti-DNP (deoxyribonucleoprotein)
Raynaud’s
Seen in SLE and drug-induced lupus
IIFA fluorescent pattern: homogenous
Anti-Sm (smith)
 Highly specific for SLE

 IIFA fluorescent pattern:
coarsely speckled
Anti-SS-A/Ro and Anti-SS-

B/La - Are also ENAs
- IIFA fluorescent pattern: finely speckled
- Substrate: HEP-2 (human epithelial)
Anti-RNP  Anti-nRNP
Centromere  Ribonucleoprotein is protein complexed

Discrete and speckled to a particular type of nuclear RNA
Antibody: Anti-centromere called U1-nRNP
Autoimmune diseases:  IIFA fluorescent pattern:
Milder form of scleroderma- coarsely speckled
CREST SYNDROME
Infrequent in:
Method Antigen source SLE Sensitivity and use

Immunofluorescenc Tissue sections and Sensitive assay- used

 MCTD
IMMUNOLOGY AND
e microscopy -chronic,elllinessymmetric andforeosivescreeningarthritis

Double ofTissueperipheralandcelljoints Precipitin reaction-
immunodiffusion -progressextracts of disease varieshigh specificity but not
(ouchterlony) decline in functional abilityveryandsensitivereduced
Counterimmunoelec life expectancy
Tissue and cell
Increased sensitivity
trophoresis extracts and speed as compared
Etiology of RA with immunodiffusion
procedure
Immunoblotting, -associated with HLA -DRB1 alleles/
Cell extracts Very sensitive- permits
western blot PTPN22 gene polymorphisms

detection of abs against
- HLA ASSOCIATION: HLA-DR4
soluble and insoluble
-Rheumatoid factor (RF) positive
antigens
Dot blot, linear blot -cigarette smoking Qualitative assay-
Purified native or
- primary cause is unknown
recombinant average sensitivity
antigens
ELISA Criteria Purified native or
for rheumatoid arthritis:
Very sensitive,
recombinant quantitative- high
antigens throughout and can
Morning stiffness
determine abs class
Arthritis in 3 or more joints areas
Arthritis of hand joints and low cost

Microsphere Purified native or Very sensitive
Symmetric arthritis
multiplexed assay recombinant (compare to ELISA),
Subcutaneous nodule
antigens semiquantitative, rapid,
Posture test for rheumatoid factor
expensive technology
Radiograph evidence of erosions in the
joints
Immunopathology of RA
Antiphospholipid Antibodies
-inflammatory process, destruction of
-heterogeneous group of antibodies bone and cartilage
-binds to phospholipids/ phospholipids
complexed with protein  Lesions in rheumatoid joints
-deep-vein, arterial thrombosis and -increase of cells lining synovial
recurrent pregnancy loss
membrane, pannus formation
-60% of lupus patients, associated with
-continual inflammation
other disease states
IL-1 , IL-6, IL-17 and TNF alpha
-identified through ability of causing false-
positive results in nontreponemal tests, lupus  Local bone erosion
anticoagulant assay and immunoassays
-over activation of osteoclasts
Lupus anticoagulant
-TNF alpha and RANKL
-a type of antiphospholipid antibody
 Autoantibodies in RA
-named due to production of APTT and PT
-affected platelet function and Rheumatoid factor (RF)
thrombocytopenia
-if suspected, factor assays will be used to -increase macrophage activity and
rule out factor deficiencies or factor specific enhance antigen presentation to T cells
inhibitors autoantibody directed against Fc portion of
IgG
isotype: IgM
Rheumatoid Arthritis
 Anticyclic citrullinated peptide
-0.5 to 1% of adult population antibody [anti-CCP or CPA]
-ages 25-55
-women 3 times as likely to be affected -provokes immune response in individuals with
than men, peaks at 65+ certain HLA-DRB1 alleles
also known as Antifillagrin antibodies
IMMUNOLOGY AND
 How both acts in RA
-after combining with specified antigen,

-key therapies focus on blocking TNF-alpha
immune complexes form and deposit in joints
-results in immune complex 2 categories of Therapy for RA
hypersensitivity reaction.
-classical complement cascade activates  Monoclonal antibodies to TNF alpha
-chronic inflammation  TNF alpha receptors fused to an IgG
Clinical signs and symptoms molecule
Laboratory diagnosis of Rheumatoid Arthritis
-joints, tendons, and bursae -combination of clinical manifestations,

-malaise, fatigue , fever, weight loss, and radiographic findings, and lab testing.
transient joint pain at small joints of hands
and feet RF used in initial diagnosis
-progress to large joints, pain causes
muscle spasms and limit movement -approx. 70-90% of patients with RA are
-may result in permanent joint dysfunction positive for RF
and deformity
-RF is present in 5% of healthy
 Osteoporosis individuals, 10-25% in ages over 65
-20 -30% of RA patients
-extra-articular manifestation development -SLE, Sjogren's syndrome, scleroderma,
mixed connective tissue disease and
 Secondary Sjogren's syndrome chronic infections
-dry eyes and mouth
-10 %  Charcoal coated IgG / latex coated IgG
manual agglutination tests used to detect
 Felty’s Syndrome RF in the past
-small percentage
-combination of chronic nodular RA -only detects IgM isotype, 75%
coupled with neutropenia and
splenomegaly and possibly -replaced by ELISA, chemiluminescence
thrombocytopenia immunoassay, and nephelometric
methods.
 Cardiovascular disease
- most common cause of death in RA -IgG and IgA tests increases test
specificity, helps in differential diagnosis
Diagnostic Classification of RA
-IgM RF levels decrease with clinical
-ACR in 1987 response to therapy
-revised in 210 with European League
against Rheumatism -elevation of IgA early = worse prognosis
-based on number of joints involved,
duration, RF and anti-CCP results, level of
ACP, CRP, ESR
TREATMENT:
Anti-CCP Testing
-nonsteroidal anti-inflammatory drugs
(NSAIDS) -ELISA, using circular synthetic form of
-Disease-modifying anti-rheumatic drugs citrullinated peptides
(DMARDS)
*methotrexate - better marker for early disease, 20-30%
present in RF- negative patients
 Biological Agents
-revolutionized RA treatment -associated with likelihood of clinically
significant disease activity
IMMUNOLOGY AND
-specificity is higher, 91-93%
Progress of Disease markers
-ESR, CRP and complement components

-suppression of inflammatory response
Granulomatosis with Polyangiitis (Wegener’s -prednisone, cyclophosphamide -rituximab
Granulomatosis) infections are the main cause of death in 50%
of GPA patients
-rare autoimmune disease
-vasculitis Immunosuppressive therapy
-localized inflammation of upper and lower
-improved patient outcomes, as much as 80%
respiratory tract
-fever, malaise, arthralgias, anorexia, and Clinical Criteria and Lab diagnosis
weight loss
majority progress to develop systemic form -WG/ GPA positive if 2 are met:
of the disease that can affect any organ
system.  nasal/ oral inflammation with oral ulcers or
purulent/bloody nasal discharge
Symptoms of Upper airway
 abnormal chest x-ray: nodules, fixed
infiltrates, cavities
rhinorrhea
 urinary sediment with microhematuria
Rhinitis Sinusitis  granulomatous inflammation on biopsy
oral/nasal ulcers  ANCA result is useful for clinicians
gingivitis
ANTINEUTROPHIL CYTOPLASMIC
Unknown etiology ANTIBODIES (ANCAs)
-multiple involved genes
 HLA-DPB1*0401- Caucasian  ANCAs are autoantibodies that are produced
 HLA-DRB1*0901- Asian against proteins that are present in the neutrophil
 HLA-DRB*1501- African American granules.
 Strongly associated with autoimmune syndromes
Staphylococcus Aureus involving vasculitis known as
 associated with relapse of WG ANCA-associated vasculitides (AAV).
These AAVs include:
PR3 o Granulomatosis with polyangiitis
-found in azurophilic granules of (GPA; Wegener’s granulomatisis)
neutrophils o Microscopic polyangiitis (MPA)
o Eosinophilic granulomatosis with
Antibody to PR3 antigen: polyangiitis (EGPA; Churg-
Strauss syndrome)
-systemic vasculitis  HOW TO TEST FOR ANCA:
-chronic infection results in TNF alpha INDIRECT IMMUNOFLUORESCENCE
release (IIF)
-stimulates neutrophils, migration of PR3 ANCA is routinely detected by IIF
from granules to neutrophil membrane using ethanol- or formalin-fixed
-binding of PR3 antibodies to PR3 antigen leukocytes as the cellular substrate.
 activation of neutrophils This method can result in either two patterns of
-release reactive oxygen species and proteolytic fluorescence:
enzymes =damage of vascular endothelium
-chronic activation of T cells induce  Cytoplasmic (c-ANCA)
differentiation of autoreactive B cells into
plasma cells, producing antibodies to PR3 Primarily caused by PR3 (proteinase 3) antigen
antigen , repeat cycle and appears as a diffuse, granular staining in the
cytoplasm of the neutrophils.
GPA therapy
IMMUNOLOGY AND
gradually fades at the outer edges of the
cytoplasm
 Perinuclear (p-ANCA)
Pattern is characterized by smooth or

homogenous staining of the multi-lobed
nuclei.
This is because the p-ANCA is caused by
antibodies against positively charged
antigens such as MPO (myeloperoxidase)
that migrate out of the granules after
ethanol fixation.
ANCA detection by IIF

has a sensitivity of more than
90% for the AAV, but has a
specificity of 80% or less because
ANCAs, especially those
producing the p-ANCA pattern,
can be found in other conditions.
Staining is most intense in the center of
the cell between the nuclear lobes and
IMMUNOLOGY AND
AUTOIMMUNTIY / GROUP 3 / BSMT 2B Samples that are positive
through initial testing with IIF
should be confirmed by
ELISA ELISA.
PR3-specific and MPO-specific
immunoassay
o Detects PR3 (c-ANCA) and MPO
(p-ANCA) antigens
In Graves disease, modifications in the
TSH receptor gene may allow the immune
system to recognize the receptor and
Follicles are filled with a material called colloid produce antibodies against it.
that has a primary constituent of thyroglobulin Environmental triggers of AITDs
(Tg) – a large iodinated glycoprotein from which include:
the active thyroid hormone triiodothyronine (T3)
and thyroxine (T4) are synthesized. Infections
 ETIOLOGY Certain medications
Smoking
Hashimoto’s thyroiditis  HLA-DR3, DR4, Psychological stress
DR5, DQ7 Pregnancy
High iodine intake and development
Graves disease  HLA-DR3
of Hashimoto’s disease  strongest
A unique feature of both Hashimoto’s and Graves is
that HLA-DR antigens are expressed on the surface link
of thyroid epithelial cells.  CLINICAL SIGNS AND
IMMMUNOPATHOLOGY
HASHIMOTO’S THYROIDITIS
ORGAN-SPECIFIC AUTOIMMUNE Also known as chronic
DISEASES lymphocytic thyroiditis
Discovered in Japan in 1912 by
AUTOIMMUNE THYROID DISEASES Dr. Hakaru Hashimoto
(AITDs) Considered to be the most common
 Diseases of the thyroid gland due to autoimmunity in autoimmune disease, affecting about
which the person’s immune system attacks and 8 out of 1,000 individuals.
damages their thyroid; interferes with thyroid Most often seen in middle-aged
function. women; women are 5-10x more likely
 The most notable AITDs are Hashimoto’s to develop the disease than men.
thyroiditis and Graves disease. Patients develop a goiter (an enlarged
thyroid) and produce thyroid-specific
 THYROID GLAND
autoantibodies and cytotoxic T cells.
located in the anterior region of the neck, Immune destruction of the
between 12-20 grams in size.
thyroid gland results in
Consists of units called follicles that are
spherical in shape and lined with cuboidal
epithelial cells.
IMMUNOLOGY AND
AUTOIMMUNTIY / GROUP 3 / BSMT 2B Characterized by
HYPERTHYROIDISM 
state of excessive thyroid
HYPOTHYROIDISM 
function
decreased thyroid function
Clinical symptoms:
Symptoms: dry skin, decreased
nervousness, insomnia,
sweating, puffy face, with
depression, weight loss,
edematous eyelids, pallor with
heat intolerance,
yellow tinge, weight gain, fatigue,
sweating, rapid heartbeat,
dry and brittle hair
palpitations,
breathlessness, fatigue,
Classic form  thyroid shows
cardiac dysrhythmias,
hyperplasia with an increased number restlessness.
of lymphocytes.
Exophthalmos 
Autoantibodies  another sign present in
Antithyroglobulin (anti-Tg) and ≈35% of patients;
Antithyroid peroxidase (anti-TPO) hypertrophy of the eye
muscles and increased
connective tissue in the
GRAVES DISEASE orbit cause the eyeball to
bulge out so that the
patient has a large-eyed staring
expression; bulging eyes
antibodies (TRAbs),
One of the most frequently occurring Antithyroglobulin (anti-Tg),
autoimmune diseases and the most and Antithyroid peroxidase
common cause of hyperthyroidism. (anti-TPO)
Women is more susceptible by a TREATMENT OF AITDs
margin of about 5 to 1 and most
HASHIMOTO’S THYROIDITIS Daily
present in the ages 50s and 60s.
Manifested as thyrotoxicosis, or an oral thyroid hormone
excess of thyroid hormones, with a replacement therapy, with
diffusely enlarged goiter that is firm levothyroxine (T4) being the
instead of rubbery. preferred drug.
Autoantibodies  thyroid- TSH levels should be monitored and
stimulating hormone receptor are used to adjust the dose of the drug.
GRAVES DISEASE
US  first line of treatment

involves radioactive iodine
Europe  antithyroid medications
with beta blocker as adjuvant therapy,
followed by continued drug treatment,
radioactive iodine therapy, or surgery
to remove part of the thyroid.
Surgery is recommended for
patients resistant to drug treatment,
but can damage the laryngeal nerves
and cause permanent hoarseness
LABORATORY DIAGNOSIS OF
AITDs
Initial screening for AITDs involves

measurement of circulating TSH levels.
TSH is routinely measured with
highly sensitive chemiluminescent
immunoassays that can detect fewer
than 0.1 mU/L.
A normal TSH level indicates normal
thyroid function; if abnormally high
or low, laboratory tests for circulating
thyroid hormone levels must be
performed.
Today, these antibodies are most
commonly detected by sensitive
ELISA and chemiluminescent
immunoassays.
HASHIMOTO’S
IMMUNOLOGY AND
AUTOIMMUNTIY / GROUP 3 / BSMT 2B than TPO antibodies in healthy persons;
since not found in all patients, a
negative result does not necessary rule
Antibodies to TPO are the best out Hashimoto’s disease.
indicator for the disease since they are
found in 95% of patients with GRAVES
Hashimoto’s disease, but only 10-15%
of the general population. Patients with Graves disease
Antibodies to Tg are less sensitive characteristically have low or
and specific because they are detected undetectable levels of TSH and
in only 60-80% of patients with the increased levels of Free T4 (FT4).
disease and are found more frequently
Antibodies to TPO and Tg are found in
the majority of patients, but are
generally not useful in making the
diagnosis. These assays can distinguish between TRAbs with
TRAbs are highly indicative of stimulatory activity versus those with inhibitory activity
Graves disease because they are because they are functional assays
present is 98-100% of patients;
therefore, included as one component
of the diagnostic criteria for Graves
disease.
TYPE 1 DIABETES MELLITUS (T1D)
o Two types of test for TRAbs:
BINDING ASSAYS  a
labeled TRAb reagent Diabetes mellitus
competes with the patient is a group of common endocrine disorders
antibody for TSH receptor
bound to a solid phase characterized by hyperglycemia (a high level of
glucose in the blood)
BIOASSAYS  require tissue
culture and thus are difficult to Three main categories:
perform; current bioassays can -classified by the American Diabetes Association
detect the ability of TRAbs to (ADA)
binf to TSH receptors on live type 1 diabetes
cells and trigger cAMP- type 2 diabetes
dependent luciferase activity. gestational diabetes.
TYPE 2 DIABETES
-characterized by insulin resistance
GESTATIONAL DIABETES
-develops in some women during
pregnancy
TYPE 1 DIABETES MELLITUS (T1D)

-characterized by a complete or nearly
complete deficiency in insulin
is a chronic autoimmune disease that involves

selective destruction of the beta cells of the
pancreas.
Can results:
insufficient insulin production

Hyperglycemia
toxic effects on the body
Long-term effects
cardiovascular complications, renal

disease,
nerve damage,
blindness, and
Infections of the lower extremities= can lead
to amputation
IMMUNOLOGY AND
Fundamental immune abnormality: failure of self-tolerance
in T cells
islets of Langerhans= responsible for the production and
secretion of the hormone, insulin
Insulin- plays a vital role in regulating the amount of glucose
Immunopathology
Autoimmune attack directed against specialized insulin-

producing cells (beta cells) that are located in spherical
clusters, called the islets of Langerhans,scattered
throughout the pancreas.
Activated CTLs migrate into an islet and begin to attack
the insulin producing cells, causing insulitis.
This is is followed by cytokine (IFN-γ, TNF- α and IL-
1) release and the presence of auto-antibodies, which
leads to a cell-mediated DTH response.
The subsequent beta-cell destruction is thought to be
mediated by cytokines released during the DTH
response and by lytic enzymes released from the
activated macrophages.
Treatment:
 Daily injectable insulin has been the

mainstay of therapy for T1D
 Immunosuppressive drugs and Biological
agents
- inhibit the autoimmune responses
that lead to beta cell destruction
 Transplantation of pancreatic beta islet
cells
IMMUNOLOGY AND
Laboratory Diagnosis of Type 1 Diabetes
Mellitus
used for T1D patients who have poor
Four criteria to be met:
glucose control
Stem cells a fasting glucose greater than
126 mg/dL on more than one
use to produce islet cells in vitro and methods to occasion (normal value is lower
induce immunologic tolerance in recipients than 100 mg/dL)
a random plasma glucose
level of 200 mg/dL or
more with classic AGE
symptoms of diabetes
Celiac disease can develop at any age after people start
an oral glucose tolerance test of eating foods or medications that contain gluten.
200 mg/dL or more in a 2-hour
 CAUSE
sample with a 75 g glucose
load; It tends to run in families and might be linked to certain
genes. Stressful medical events such as a viral infection
or surgery can trigger it. So can emotional trauma or
pregnancy. If one of your close family members has it,
like a parent or sibling, you have a 1 in 10 chance of
getting celiac disease.
SYMPTOMS INCLUDE:
 Headache
 Nausea
 Weight loss
 Fatigue
 Gastrointestinal distress
 Bloating
 Stomach pain
 Diarrhea
TREATMENT
The only treatment for celiac disease is to follow a
a hemoglobin A1c value
gluten-free diet that is, to avoid all foods that contain
(HbA1c) greater than 6.5% gluten.
GAD and IA-2A- test for antibodies to confirm the
diagnosis if T1D is suspected
AUTOIMMUNE LIVER DISEASE
Western blotting, ELISA, and mass
spectrometry
2. used to detect antibodies to other The liver is central to destruction and
pancreatic antigens (insulin, GAD, and IA-2) detoxification:
Combined screening for IA-2A, ICA, and GAD It is the largest gland in the body
antibodies Weighs 3-4 pounds
 have the most sensitivity and best positive Has two lobes (right and left lobe)
It is the only organ that can regenerate,
predictive value for T1D in high risk populations
meaning if there are some damage or some
parts that are removed after a surgery it can
regenerate back to its original size.
CELIAC DISEASE
In addition, the liver helps the body digest food,
also known as gluten-sensitive store energy, makes proteins, protects the body
enteropathy against foreign invaders and remove toxins.
is a digestive and autoimmune disorder that Bile is created in the liver and is transported to
results in damage to the lining of the small the gallbladder for storage via the bile ducts.
intestine when foods with gluten are eaten.
IMMUNOLOGY AND
AUTOIMMUNTIY / GROUP 3 / BSMT 2B B. AUTOIMMUNE HEPATITIS
C. PRIMARY SCLEROSING CHOLANGITIS (PSC)

OUTLINE
A. PRIMARY BILIARY CIRRHOSIS (PBC)

PRIMARY BILIARY CIRRHOSIS (PBC):
INTRODUCTION
PBC: DIAGNOSIS
Leads to progressive destruction of the
intrahepatic bile ducts that can lead to cirrhosis. For the diagnostic criteria you only need to meet two of
Leads to: the three following :
chronic cholestasis (flow of bile is
slowed or blocked) Elevated alkaline phosphatase greater than 1.5
inflammation of the portal vein In the times the upper limit of normal
liver A positive AMA greater than or equal to 1 to 40
accumulation of scar tissue that can Liver biopsy – can confirm the diagnosis of PBC;
ultimately lead to cirrhosis and however, a person does not always need this test.
eventually liver failure. A health care provider will only perform a biopsy
Target cells or tissues: intrahepatic bile if the AMA blood test is negative and the person
ducts. shows other signs of PBC.
Most common autoimmune liver PBC: TREATMENT

disease 3. Ursodeoxycholic acid (UDCA) – a bile acid that
90% are women helps move bile through the liver. This
medication can improved histology meaning the
medium age at diagnosis is 50s liver inflammation and survival overall in patients
with PBC and it is a weight based regimen.
the hallmark of this disease is a
4. new FDA approved medication known as
positive Anti-mitochondrial antibody (AMA).
Obeticholic acid can be added if there is an
inadequate response to UDCA.
5. However if the disease advances patients are at
PBC: SYMPTOMS risk for development of cirrhosis or liver cancer.
In terms of symptoms, most people are  AUTOIMMUNE HEPATITIS:
asymptomatic. Primary care doctors are INTRODUCTION
checking liver test annually and the first sign is
isolated elevation in alkaline phosphatase Formerly known as active chronic
(ALKP) hepatitis.
Symptoms if present usually includes pruritus immune-mediated liver disease that can lead
to end-stage liver failure without treatment
(itchy skin), fatigue, dry eyes and mouth,
target cell: liver
abdominal pain associated autoantibodies for two types of
Some people may also have jaundice (a AIH:
condition that causes the skin and whites of the For AIH-1: characterized by presence of
eyes to turn yellow. Smooth muscle antibodies; ANAs
For AIH-2: characterized by the detection of
specific anti-liver/kidney microsomal
antibody type 1(anti-LK M-1) or infrequently
anti-LKM type 3 (anti-LKM3) and/or antibodies
against Anti-liver cytosol type 1 antigen (anti-LC-
1).
4. more common in females.
5.The hallmark of this disease is that it
responds to prednisone.
IMMUNOLOGY AND
AUTOIMMUNTIY / GROUP 3 / BSMT 2B weight loss, abdominal pain, itching, and
maculopapular rashes.
AIH: SYMPTOMS  Patients have symptoms of portal
hypertension such as gastrointestinal
 The clinical feature of AIH can be quiet bleeding or hypersplenism.
variable.  Jaundice may also be present.
 25% are asymptomatic.
AIH: LABORATORY FINDINGS
 Adults usually present with an unexpected onset
of vague symptoms such as fatigue, nausea,
 Common laboratory findings include:
Elevated alanine aminotransferase (ALT),
aspartate aminotransferase (AST), with lest C. PRIMARY SCLEROSING CHOLANGITIS (PSC)
prominent increases in serum bilirubin, and
alkaline phosphatase. autoimmune destruction of the bile ducts.
Serum immunoglobulin levels, it mostly affect men, 30-40 years of age.
particularly IgG, are high all patients with PSC must undergo
And various autoantibodies are also present colonoscopy with biopsies.
such as the ANAs, ANCAs, Smooth muscle -Mostly asymptomatic to pruritus and
cholangitis
antibodies (SMA), anti-liver/kidney
some may also present fatigue and
microsomal antibody type 1(anti-LKM-1),
jaundice.
Anti-liver cytosol type 1 antigen (anti-LC-1), they have hallmark elevated alkaline
and anti-mitochondrial antibodies (AMAs) phosphatase
AIH: TREATMENT Diagnosis is made with MRI where you can
see abnormal bile duct.
Treatment of autoimmune hepatitis is based
on severity of the disease PSC: TREATMENT
The mainstay of treatment is suppressing the
immune system and done initially with steroids 2. Treatment options are limited and mainly
commonly prednisone, also Azathioprine, or supportive
Mycophenolate if patients cannot tolerate 3. No FDA approved regimen.
azathioprine. 4. It is very important to have a colonoscopy at the
Patient duration is atleast 2 years after time of diagnosis to evaluate for inflammatory
normalization of liver tests, however due to risk bowel disease. If patients have inflammatory
of relapse treatment might be indefinite. bowel disease the risk of colon cancer is much
If the disease advances despite treatment higher.
patients are at risk for cirrhosis or liver cancer. 5. If the disease does progress. Patients are at risk
If untreated, AIH usually progresses to liver for cirrhosis or liver cancer.
failure, at which point liver transplantation is 6. Other complications of PSC include: bacterial
required. cholangitis, colon cancer, and at high risk for
- gallbladder cancer and bile duct cancer known
as cholangiocarcinoma.
MULTIPLE SCLEROSIS
INTRODUCTION
 Multiple sclerosis is an autoimmune disorder

involving inflammation of the Central
Nervous System.
 It is the demyelinating disease of the CNS which
include the brain and the spinal chord.
 It is characterized by the formation of lesions
called plaques in white matter of the brain and
spinal chord, resulting in the progressive
destruction the myelin sheath of axons.
 The T cells and macrophages within these
plaques orchestrates demyelination. So the
hallmark of MS is demyelination. We know that
myelin is the protective sheath that surround the
axons of neurons
IMMUNOLOGY AND
AUTOIMMUNTIY / GROUP 3 / BSMT 2B MS demyelination happens when the immune
system inappropriately attacks and destroys the
myelin, which makes communications between
allowing them to quickly send electrical signals.
neurons break down leading to all sorts of
Myelin sheath are produced by oligodendrocytes,
sensory, motor, and cognitive problems.
which are group of cells that support neurons. In
 MS is most closely associated with inheritance
of a particular HLA molecule coding for the
beta chain of the DR subregion, namely the
The cause is unknown, however it is linked to
DRB1
both genetic and environmental factors.
 Inflammatory response is triggered by
Genetic Risk Factors include: being female, and
mimicry
having genes that encode for a specific type of
 It affects 20-40 years old.
immune molecule called HLA-DR2 which is
 The associated autoantibodies: antibodies to used to identify and bind to foreign molecules.
myelin basic proteins Environmental risk factors might include
 The target cells or tissues: myelin sheath of
reduced exposure to sunlight, vitamin D
nerves. deficiency and cigarette smoking
MS: DIAGNOSIS
3. The diagnosis of MS is supported by MRI which

shows multiple central nervous lesions, called
the white matter plaques.
4. Lumbar puncture
5. Evoked potential can also be helpful because it
measures the nervous system’s response to
visual stimuli.
MS: TREATMENT
MS: LABORATORY TESTS:  There is no cure for multiple sclerosis

 However, there are medications which are
Oligoclonal Banding
particularly effective for the relapsing-remitting
4. Distinct bands on CSF protein type because they lessen the severity of relapses
electrophoresis that are not seen in the serum and make them happen less frequently. These
5. Identified in more than 90% of the MS medications include Corticosteroids,
patients cyclophosphamide, intravenous immunoglobulin.
6. However it is not diagnostically specific.
 Chronic treatment for MS includes
MS: SYMPTOMS immunosuppressants like recombinant beta-
IFN which decreases the level of inflammatory
Damage to the tissue of the CNS can cause visual cytokines in the brain and increases the
disturbances, weakness or diminished dexterity in function of T regulatory cells.
one or more limbs, locomotor incoordination,
dizziness, facial palsy, and numerous sensory
abnormalities such as tingling or "pins and
Myasthenia Gravis
needles" that run down the spine or extremities
An autoimmune disease that affects the
and flashes of light seen on eye movement.
neuromuscular junctions.
50 to 70 percent of patients alternate between It is characterized by weakness and
remissions and relapses for many years. fatigability of skeletal muscles.
The pattern of the disease is heterogeneous in
remainder follow a chronic progressive course. terms of its age of onset and gender
involvement.
MS: CAUSE
Myasthenia gravis is caused
- by an abnormal immune reaction (antibody-
mediated autoimmune response) in which the
body's immune defenses (i.e., antibodies)
IMMUNOLOGY AND
inappropriately attack certain proteins in muscles

that receive nerve impulses.
In MG, antibody-mediated damage to the Late onset MG

acetylcholine receptors in the skeletal Early onset MG
muscles or to other proteins in the  Occurs before the age  Occurs after the
neuromuscular junctions leads to of 40. age of 40
progressive muscle weakness.  Affects  Affects
-Caused by an error in the transmission of nerve predominantly predominantly
impulses to muscles. It occurs when normal females males
communication between the nerve and muscle is  Women >men  Men>women
interrupted at the neuromuscular junction—the  HLA association:  HLA association:
place where nerve cells connect with the muscles  HLA A1  HLA B7
they control.  HLA B8  HLA DR2
 HLA DR3
There are two other antibodies that cause the other  The thymus also  About 10%-15% of
15% of cases of MG. These are antibodies against appears to play a role patients have a
muscle-specific kinase (MuSK) and low density in the autoimmune thymoma, it is a
lipoprotein receptor-related protein 4 (LRP4). process of MG. tumor of the
MuSK and LRP4 are important proteins for the Thymic hyperplasia is thymus that may
creation and organization of the ACHR. common in EOMG contain auto
Destruction of these proteins by autoantibodies patients with ACHR reactive T cells. It
leads inadequate ACHR. This causes the symptoms antibodies. The is thought that
of MG. follicles in the thymus inflammation of
expand and contain the thymus gland
The severity of symptoms can vary dramatically ectopic germinal may be triggered
between patients. They can be mild and subtle or centers with by persistent
life threateningly severe involves the facial, bulbar autoantibodies- activation of TLRs
and respiratory muscles. producing B cells. by viruses such as
Symptoms most affect the proximal muscles and EBV.
small muscle of the head and neck.
Early signs Antibodies
Ptosis (drooping of the eyelids) Acetylcholine receptor (ACHR) – 80%-85% of the
– eyelid muscle weakness patients
Diplopia (double vision)
-because of the extra ocular ACHR, which appears to contribute to the
muscle weakness pathogenesis of the disease by three mechanisms.
Inability to retract the corners of the mouth Normally, ACH is release from nerve endings to
(snarling appearance) generate an action potential that causes the muscle
Muscle weakness (upper limbs) fiber to contract. When the antibody combines with
Difficulty in speaking, chewing, and the receptor site, binding of ACH is thought to be
swallowing, and inability to maintain blocked.
support of the trunk, neck, or head. – facial
muscle weakness Muscle-specific kinase (MuSK) – 4% of the
Respiratory muscle weakness patients, mostly young females
– it can be life threatening
Patients lacking anti-ACHR may produce antibodies
to other proteins involved in the neuromuscular
junction. Muscle-specific kinase (MuSK), it is an
enzyme that plays an important role in the
development of the neuromuscular junction and in the
clustering of ACHRs on the muscle cell membrane,
which enhances the transmission of the signals from
the nerve cells.
Lipoprotein receptor-related protein 4 – 2% of

patients with generalized MG
IMMUNOLOGY AND
AUTOIMMUNTIY / GROUP 3 / BSMT 2B with LRP4 antibodies have a mild form of MG.
These patients are typically young females.
LRP4, a lipoprotein involved in the activation of Mechanism of the immunologic injury in MG Motor
MuSK. It can also be used to monitor disease neurons
severity. Lipoprotein receptor-related protein 4 sends signals for muscle contraction.
(LRP4) antibodies are found in up to 50% of
It is distributed all over the body making
patients who are negative for both AChR and MuSK
contact with muscle which are present in the
antibodies. People
various part of body.
Neuromuscular junction
contact point of motor neuron and muscle.
Neurons
The enzyme
 first component of the neuromuscular
junction  acetylcholinesterase will then removes and
degrade ACH from the muscle receptors. But in
 innervate (to supply with nerves) skeletal muscle
most types of MG, IgG antibodies is attach to the
contain vesicles full of the neurotransmitter
ACHRs and block ACH binding sites.
ACH.
The MuSK
 When this motor neuron receives the signal, it
releases ACH molecules in the NMJ. So here,
the ACH molecules bind to ACHR. In response  an intra-membrane enzyme that is typically
to this binding, the muscle cells get contracted responsible for destroying old or faulty ACH-
and we are able to carry out different functions gated sodium channels in healthy cells.
like walking, talking, breathing and even it helps  In MG, the MuSK protein recognizes
to keep open our eyelids. malfunctioning IgG bound ACHR and
signals the muscle membrane to internalize
 Across the synaptic cleft lies a muscle cell, it them into vesicles.
has a lot of deep grove in its membrane that  They are subsequently destroyed so that they can be
increase its surface area which is prepared replaced by functioning receptors. This type is
with ACHR. Each receptor is actually a cause by antibodies that target the MuSK protein or
sodium channel made up of five subunits. other unidentified postsynaptic targets and accounts
for the remaining 15% of cases.
 While ACH is not bound, it remains in the closed
position.
In MG, the patient’s B-cells produces antibodies
 At the neurons, sodium influx has triggered against ACHR that is anti-ACHR antibodies.
ACH vesicles to bind and merged with the Normally, B-cells don’t produce antibodies
cell membrane-this allows vesicles to dump against our own molecule that is the self-
their ACH into the synaptic cleft. antigens. But in this case, antibodies are
produced against the self-molecules such
 ACH then diffuses across the cleft and binds to conditions are called as autoimmune diseases
ACHRs. This opens the receptor channels and and antibodies involved are called
allows sodium to rush into the muscle cell. – So autoantibodies. Once antibodies are produced,
this triggers the muscle movement. they binds to their target that is ACHR.
Treatment
 Anticholinesterase agents for main therapy.

used for the main therapy and
anticholinesterase agents to prevent the
destruction of the neurotransmitter.
We can also use the acetylcholinesterase
inhibitors usually pyridostigmine and
neostigmine, and these increase the amount
of the ACH at the neuromuscular junction
and improve the sypmtoms.
 Thymectomy for patients who have a thymoma. If
these treatment is not effective, then ,
Immunosuppression is
recommended.
IMMUNOLOGY AND
AUTOIMMUNTIY / GROUP 3 / BSMT 2B suppress the production of antibodies and
improved the condition.
 Plasmapheresis or intravenous
 Generally, begins with high dose of corticosteroid
immunoglobulin can be administered to
drugs followed by another immunosuppressive
patients in crisis.
drug such as:
Plasmapheresis a.k.a plasma exchange
Azathioprine or mycophenolate mofetil to
maintain the response. And also it can
-This process removes harmful antibodies form
the blood, which may result in an improvement
in muscle strength.
-It is a short-term treatment. The body continues performing the steps of the RIPA. This method
to produce the harmful antibodies and weakness increases the likelihood of detecting antibodies
may recur. Plasma exchange is helpful before present in low concentration.
surgery or during time of extreme MG weakness.
Alternative methods:
Intravenous immunoglobulin (IVIG)
 is a blood product that comes from donors. It  Immunofloresence cell based assays in which
is used to treat autoimmune MG. Although it patients serum is incubated with HEK293 cells
is not entirely known how IVIG works, it expressing all four ACHR SUBUNITS. This
affects the creation and function of highly sensitive assay can detect antibodies toward
antibodies. ACHR clusters in patients that were previously
classified as seronegative by RIPA.
Others assays that have been developed

includes:
 ELISA
 Luciferase immunoprecipitation
 FIPA – fluorescence immuoprecipitation assay uses
ACHR subunits or MuSK antigens labeled with green
fluorescent protein to detect patient antibodies and has
a sensitivity that is similar to RIPA.
Goodpasture’s Syndrome
 Biological agents:
- monoclonal antibodies or fusion proteins  A life threatening autoimmune disease
targeted to specific components of the immune system characterized by diffuse pulmonary
involved in the pathogenesis of MG offer hope to MG hemorrhage and glomerulonephritis.
patients who are unresponsive to conventional  Injury is mediated by the production of
therapies. autoantibodies to a component of the glomerular
basement membrane known as anti-GBM
Laboratory diagnosis of MG antibodies.
 Originally identified – Ernest
 Radioimmunoprecipitation assay (RIPA)– most Goodpasture in 1919.
commonly used procedure for antibody to the  Found mainly in Caucasian of European
ACHR, which is based on precipitation of the origin.
patient’s antibody with ACHRs isolated from
human muscle. It primarily affects two age groups:
 Men – 30s
 The complex is detected with a radio-labeled snake  Men and women – 60s and 70s
venom called α-bungarotoxin which binds with  Characterized by the presence of autoantibodies
high affinity to a different site on the receptors. to antigen in the basement membranes in the
This assay is sensitive and can be used to determine glumeruli of the kidneys and alveoli of the lungs.
the antibody titers.  Most patients initially experience fatigue and
malaise followed by clinical signs of kidney
 Similar RIPA method using I-labeled human involment:
MuSK is used to detect antibody to MuSK.  Edema
Sensitivity of the method has been increase by using  Hypertension
two-step RIPA in which antibodies are isolated by -which can rapidly progress to acute renal
an affinity purification process using Sepharose failure if left untreated.
beads containing immobilized antigen before Some patients develop
 Chronic Renal Failure – that requires
IMMUNOLOGY AND
AUTOIMMUNTIY / GROUP 3 / BSMT 2B  Kidney transplantation
So about -
 Lifetime hemodialysis
 60% to 70% of patients with GS have levels
pulmonary involvement and exhibit
symptoms such as:
 Cough
 Shortness of breath
 Hemoptysis (coughing up blood)
 More likely to occur in young males

between ages of 18 and 35
 Develops in susceptible people after
environmental exposures:
 Viral infections
 Cigarette smoke
 Expose to volatile hydrocarbons or
oxidants
Inflict enough damage to expose alveolar capillary
antigens to circulating antibodies.
Pathophysiology
 Autoantibodies produced in GS – specifically
directed against the non-collagenous domain of
the alpha-3 chain type IV collagen
 Reacts with collagen in the glomerular
or alveolar membranes and causes
damage by type II hypersensitivity.
 HLA association: 70%-80% of patients carry the
HLA-DRBI-15 antigen
Complement binding to immune deposits attracts
neutrophils, which mediates injury to the membranes by
releasing chemically reactive oxygen- containing
molecules and photolytic enzymes.
 Immune reactants progressively destroys:
 Renal tubular
 Glomerular
 Pulmonary alveolar basement
membranes
 Loss of membrane integrity results in
leakage of blood and proteins into the urine.
Renal involvement: Pulmonary

symptoms:
 Gross or  Hemorrhage
microscopic
hematuria
 Proteinuria  Dysnea
 Decrease 24-hour  Weakness
creatinine clearance
 Elevated or increase  Fatigue
in blood urea and
serum creatinine  cough
testing detects and measures the amount of these
autoantibodies in the blood.
 Between 20% and 35% of the patients are also
 Standard treatment of Goodpasture’s syndrome involves: positive for ANCAs, which are usually specific for
 administration of high dose corticosteroids – myeloperoxidase and exhibit the perinuclear staining
stop inflammation pattern of IIF.
 Followed by immunosuppressive drugs  Detectable months to years before anti-GBM and
such as: symptoms are evident.
 Cyclophosphamide – inhibit further
production of antibodies.  Patients with renal disease, these antibodies are
 Plasmapheresis is performed to remove indicated by formation of a smooth, linear,
circulating autoantibodies. bonelike pattern of fluorescence along the GBM.
 Prompt initiation of therapy is important – because  Glomerulonephritis caused by other autoimmune
symptoms can be life threatening. disease shows a granular pattern of
 If started early – can prevent renal failure immunofluorescence caused by nonspecific
 Long term prognosis is poor – leads to death deposition of immune complexes in the
glumeruli.
 Anti-neutrophil cytoplasmic antibodies
 Patients with pulmonary involvement, linear
(ANCA) are autoantibodies produced by the immune IgG staining of the alveolar cells can be seen on
system that mistakenly target and attack specific proteins direct immunofluorecence of lung biopsy tissue
within neutrophils (a type of white blood cell). ANCA or bronchial washings.
IMMUNOLOGY AND
Gold standard in ANA testing

IIF test
human epithelial cell line HEp-2 as the
SUMMARY substrate.
Main fluorescent patterns observed:
 Autoimmunity - Breakdown in self- Homogeneous

tolerance. Peripheral
Speckled Nucleolar
Multiple causes: Centromere.
Genetic
Environmental  Rheumatoid factor
Nutrition
- an autoantibody directed against the Fc
Infections
portion of IgG molecules.
Immunologic tolerance is achieved at two levels. -patients with rheumatoid arthritis
 Central tolerance- Immature self-reactive  Antineutrophil cytoplasmic antibodies

lymphocytes that recognize self antigens in (ANCAs)
generative (“central”) lymphoid organs die by
-associated with autoimmune syndromes:
apoptosis; other fates
vasculitis.
 Peripheral tolerance -mature self-reactive -IIF using ethanol- or formalin-fixed
lymphocytes that recognize self antigens in leukocytes as the substrate.
peripheral tissues are inactivated (anergy),
Two patterns of fluorescence can result:
killed (deletion) or suppressed.
 Infectious microorganisms- trigger  c-ANCA- granular staining of the
autoimmune responses cytoplasm of the neutrophils
 p-ANCA- characterized by
fluorescence surrounding the nuclear
SYSTEMIC DISEASES: lobes of ethanol-fixed neutrophils
- SLE, RA, and GPA

ORGAN- SPECIFIC DISEASE:
- Hashimoto’s thyroiditis, Graves disease, type 1
diabetes mellitus, celiac disease, autoimmune
hepatitis, primary biliary cirrhosis, multiple sclerosis,
myasthenia gravis, and Goodpasture’s syndrome
Specific antibodies
- Anti-dsDNA antibodies – SLE

- Anti-CCP (cyclic citrullinated proteins) antibodies-
RA
- Antibodies against the thyroid-stimulating hormone
receptor- Graves disease
 Antinuclear antibodies (ANAs)
- patients with SLE

-patients with other connective tissue
diseases.
IMMUNOLOGY AND
References:
Autoimmune hepatitis (n.d)

https://www.slideshare.net/pratapsagar/autoimmun
e-hepatitis-191034467
Osmosis. (n.d) Multiple sclerosis – causes,
symptoms, diagnosis, treatment, patholoy
https://youtu.be/yzH8ul5PSZ8
WCM Surgery (n.d) autoimmune liver disease
https://youtu.be/yzH8ul5PSZ8
Cutolo, M., et.al. (n. d). Sex hormones influence on
the immune system: basic and clinical aspects in
autoimmunity.
https://pubmed.ncbi.nlm.nih.gov/15485092/#:~:text
=Sex%20hormones%20seem%20to%20play,and%
20glucocorticoids)%20as%20natural%20immunos
uppressors.
Peripheral tolerance.(n.d).
https://en.wikipedia.org/wiki/Peripheral_tolerance#
:~:text=Peripheral%20tolerance%20is%20the%20s
econd,do%20not%20cause%20autoimmune%20dis
ease.
What is Self- Tolerance. (n.
d).https://www.genscript.com/self
tolerance.html#:~:text=Self%2Dtolerance%20is%2
0the%20ability,physiological%20function%20and
%20overall%20health.
IMMUNOLOGY
TUMOR IMMUNOLOGY AND
SEROLOGY (IS)
CONTENTS
I. Introduction to Tumor A. Active Immunotherapy and

Biology Cancer Vaccines
A. Classification of Tumors B. Passive Immunotherapy
1. Major Types of
Genes Involved in
Malignant
Transformation
2. Classification of
Malignant Tumors
3. The TNM System
B. Tumor Antigens
Tumor-Specific
Antigens (TSAs)
Tumor-Associated Antigens
Clinically Relevant
Tumor Markers
Clinical uses of Tumor
Markers: Benefits and
Limitations
Serum Tumor Markers
Laboratory Detection of Tumors

Tumor Morphology
Immunohistochemistry
Immunoassays for
Circulating Tumor
Markers
Molecular Methods in
Cancer Diagnosis
 Interactions Between
The Immune System and
Tumors
Immune Defenses
Against Tumor Cells
Innate Immune Response
Adaptive Immune response
IV. Immunoediting and Tumor
Escape
Elimination
Equilibrium
Escape
V. Immunotherapy
IS TUMOR IMMUNOLOGY Page

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IMMUNOLOGY
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INTRODUCTION TO TUMOR BIOLOGY
Terminologies:
Tumor immunology - is the study of the antigens associated with tumors, the immune
response to tumors, the tumor’s effect on the host’s immune status, and the use of the
immune system to help eradicate the tumor.
Tumor - it is a continuous abnormal proliferation of cells without control.
Cancer - came from the Latin word for “crab,” derives its name from this property of
invasiveness, which can resemble the legs of a crab when viewed in microscopic tissue
sections.
Today, it is recognized that cancer is not a single disease, but rather a heterogeneous group
of diseases that show variability in these characteristics. In fact, heterogeneity is
commonly found among different cancer cells in the same patient.
Apoptosis - normal homeostatic mechanism that is necessary for maintaining normal cell
numbers. This process occurs through a series of biochemical reactions within a cell that
lead to chromatin condensation, DNA fragmentation, cell shrinkage, and membrane
blebbing. Cell death occurs in the absence of cell lysis and does not provoke an
inflammatory reaction that could damage neighboring cells. Genetic changes within a cell
that cause it to become resistant to apoptosis are important in the development of cancer.
Carcinogenesis - The transformation of a cell into a malignant tumor is thought to be a
multistep process involving a series of genetic mutations that cause the phenotype of a cell
to be changed over time.
Classification of Tumors:
 Benign tumors - composed of slowly growing cells that are well differentiated and
organized, similar to the normal tissue from which they originate.
Surrounded by a capsule, which secures them in place and prevents them from
circulating to other parts of the body.
 Malignant Tumors - also known as cancer cells. These are disorganized masses that
rarely encapsulated, allowing them to invade nearby organs and destroy their normal
architecture. In addition, malignant tumors are commonly exhibit metastasis, or the
ability of cells to break away from the original tumor mass and spread through the blood
to nearby or distant sites in the body.
Major Types of Genes Involved in Malignant Transformation

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IMMUNOLOGY
SEROLOGY (IS)
 Proto-oncogenes – are normal genes involved in normal cell growth and division.
Alterations in these genes can convert them into oncogenes, which are involved in
malignant transformation
 Tumor suppressor gene – control cell division by regulating the progression of cells
through the cell cycle and maintaining genetic stability of the cells by repairing damaged
DNA.
Classification of Malignant Tumors
Carcinomas - approximately 80% of cancers, derived from the skin or epithelial linings
of internal organs or glands.
Leukemias or lymphomas - about 9%. These are malignant white blood cells (WBCs)
present in the circulation or lymphatic system.
Sarcomas - about 1%, derived from bone or soft tissues such as fat, muscles, tendons,
cartilage, nerves, and blood vessels.
FEATURE BENIGN TUMOR MALIGNANT TUMOR
Degree of Resemble the tissue of origin and Poorly or completely

differentiation are well differentiated undifferentiated
Rate of growth Slow growing Rapid/fast growing
Local Invasiveness Not invasive Invasive
Distant Remain localized to the site of Locally invasive and

spread/Metastasis origin (minimal mitotic activity) metastasize to distant sites
Staging of Cancers
To make a specific diagnosis and guide treatment decisions, physicians use staging
systems based on the site and type of the primary tumor, tumor size, involvement of
regional lymph nodes, presence or absence of metastasis, and degree of resemblance to
normal tissue.
TNM system of the American Joint Committee on Cancer (AJCC) - most widely used
staging scheme.

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IMMUNOLOGY
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THE TNM SYSTEM

In the TNM system, tumors are classified according to the size and extent of the primary
tumor (T0 to T4), the degree of spread to adjacent lymph nodes (N0 to N3), and the presence or
absence of distant metastases (M0 or M1). For example, breast cancer that is classified as
T2N1M0 would involve a tumor between 2 to 5 cm in diameter that has spread to one to three
regional lymph nodes but has not spread to distant sites.
T: Primary tumor
Tis = Carcinoma in situ
Ta = non invasive
Tx = cannot be evaluated
T0 = free of tumor
T1 = lesion < 2cm
T3 = skin and/or chest wall involved by invasion
T4 = tumor invasion/ fixation
N: Regional lymph node involvement
High number denotes increasing extent of involvement
Nx= not evaluable
N0= no axillary nodes involved
N1= 1 mobile regional (axillary) node involved
N2= multiple, mobile regional nodes involved
N3= fixed regional lymph node involved

N4= beyond regional lymph node involvement
M: Metastasis
M0= no evidence of metastases
M1= distant metastases are present

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IMMUNOLOGY
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Mx= distant metastases not evaluable
Characteristics of Cancerous Cells

In 2000, Hanahan and Weinberg defined a cancerous cell as one that exhibits six
characteristics:
 Sustained signaling of proliferation
 Resistance to cell death
 Ability to induce angiogenesis (development of new blood vessels to provide
oxygen and nutrients to the tumor)
 Immortality in terms of cell division
 Invasion and metastasis
 Ability to avoid suppressors of cell growth
In 2011, Hanahan and Weinberg proposed four additional hallmark characteristics of

cancer cells:
 Reprogramming of energy metabolism to support malignant proliferation
 Ability to evade destruction by the immune system
 Genomic instability and mutations
 Inflammatory responses that promote tumor growth
Tumor Antigens
The concept of tumor immunology is based on the premise that tumors possess antigens that are
recognized as foreign by the immune system. Tumor antigens can be broadly classified into two
groups: tumor-specific antigens (TSAs) and tumor associated antigens (TAAs).

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IMMUNOLOGY
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Tumor-Specific Antigens (TSAs)

TSAs are unique to the tumor of an individual patient or shared by a limited number of
patients with the same type of tumor.
They are coded for by viral oncogenes or by host proto-oncogenes or tumor suppressor
genes that have undergone genetic mutations.
A well-known example of a TSA is a fusion protein that is produced in chronic
myelogenous leukemia (CML) cells. This protein is a result of a reciprocal chromosome
translocation commonly known as the Philadelphia chromosome, which involves the BCR
(breakage cluster region) on chromosome 9 and the c-ABL gene on chromosome.
During the translocation, the two chromosomes break and exchange parts so that the c-ABL
gene is combined with part of the BCR to produce a hybrid gene that is constantly
expressed
originate from point mutations (i.e., genetic changes involving a single base pair) in key
genes involved in cell proliferation, such as the tumor suppressor gene.
Tumor-Associated Antigens (TAAs)
Are expressed in normal cells as well as in tumor cells. Tumor cells abnormally express
these protein or carbohydrate antigens in terms of their concentration, location, or stage of
differentiation.
Major categories of peptide TAAs

 Shared TSAs
are expressed in many tumors, but not in most normal tissues. The only normal
cells in which they have been detected are testicular germ cells (i.e., spermatogonia
and spermatocytes) and, to a lesser extent, placental trophoblasts and ovaries.
Hence, these antigens have also been called cancer/testis antigens. They become
TAAs when the transformation process causes them to be expressed on tumors
originating from other cell types. These antigens have been identified on many
tumors of epithelial or mesenchymal origin.
The best-known examples of TAAs in this category are the melanoma antigen
gene (MAGE) proteins that are expressed by melanoma tumors.
Differentiation antigens
are expressed on immature cells of a particular lineage. An example of a TAA in
this group is the CD10 antigen (previously known as the CALLA, or common
acute lymphoblastic leukemia antigen), which is normally found on pre-B cells but
not on mature B cells.
This category of antigens also includes the oncofetal or embryonic antigens that
are normally expressed on developing cells of the fetus but not on cells in the
adult. It is thought that the genes coding for these antigens are silenced during
development of the embryo, but that the process of malignant transformation
allows them to be re expressed.

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IMMUNOLOGY
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Examples of oncofetal antigens include carcinoembryonic protein (CEA), alpha-

fetoprotein (AFP), and prostate-specific antigen (PSA). Many of these antigens are tumor
markers that can be detected in the clinical laboratory (see the next section).
 Overexpressed antigen
which are found in higher levels on malignant cells than on normal cells. Genetic
mutations that occur during transformation are thought to deregulate expression of
these proteins, resulting in levels up to 100 times greater than normal. A well-
known example of a TAA in this category is the human epithelial growth factor
receptor 2 (HER2) protein, a transmembrane receptor that binds human epidermal
growth factor. Gene amplification in a certain type of breast cancer can result in
overexpression of this protein, which serves as a marker for detection and therapy
(see Immunotherapy later). In addition to peptide TAAs, glycolipid and
glycoprotein antigens may also be overexpressed in some tumors.11 Examples of
these antigens include cancer antigen 125 (CA 125), which is associated with
ovarian cancer, and cancer antigen 19-9 (CA 19-9), which is associated with
pancreatic cancer. In the next section, we will discuss clinical applications of some
of these markers.
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Clinically Relevant Tumor Markers
Tumor markers
biological substances found in increased amounts in the blood, body fluids, or tissue of
patients with a specific type of cancer
can be produced by the tumor itself or by the patient’s body in response to the tumor or
related benign conditions
Concentration of a tumor marker in the serum depends on:

degree of tumor proliferation
size of the tumor mass
proteolytic activities of the tumor
release of the marker from dying tumor cell
Elevated level of a tumor marker = significant amount of a particular type of tumor is present
Characteristics of an ideal tumor marker:
Be produced by the tumor itself or by the patient’s body in response to the tumor
Be secreted into a biological fluid, where it can be inexpensively and easily quantified

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Have a circulating half-life long enough to permit its concentration to rise with increasing
tumor load
Increase to clinically significant levels above the reference level while the disease is still
treatable
Have a high sensitivity; in other words, it should easily detect the majority of individuals in
the population who have a particular cancer
Have a high specificity; in other words, the marker should be absent from, or present at
background levels in all individuals without the malignant disease in question to minimize
false-positive test results
Clinical Uses of Tumor Markers: Benefits and Limitations

Four major clinical applications of tumor markers: screening, diagnosis, prognosis, and
monitoring
Clinical uses of tumor markers:
 To screen asymptomatic individuals in a population for the presence of cancer
The individuals screened may belong to a healthy population or to a high-risk population.

For example, PSA has been routinely used to screen men aged 50 or older for the presence
of prostate cancer.
 can be helpful in making an initial diagnosis of cancer
For example, an elevated level of PSA might suggest the presence of prostate cancer

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third use of tumor markers is in predicting prognosis of a cancer patient
In general, an initial high concentration of a tumor marker or an increasing level of a

tumor marker over time points to a poorer prognosis
 to monitor known cancer patients to determine whether their treatment is
working and to check for recurrence of their tumor
Decreasing levels indicate that the treatment is effective in reducing the amount of tumor
harbored by the patient, whereas increasing levels indicate that the treatment is ineffective
and that the number of tumor cells in the patient is increasing.
Screening of Tumor Markers
detection of tumor markers provides an ideal way to screen for tumors because the
markers can be detected by a simple blood test
benefits and limitations of using a particular marker to screen a population must be
considered before testing is implemented
Advantages of screening for tumor markers:
screening asymptomatic individuals can lead to earlier detection of tumors with a need for
less aggressive treatment
In addition, many people who have been screened can receive reassurance from true
negative results.
Disadvantages:
Besides the actual dollar costs of the screening test, harm to the individuals tested may
also occur
Misleading reassurance can be experienced by individuals with false-negative results. In
contrast, false-positive results can lead to patient anxiety; possible harm from more invasive,
unnecessary follow-up testing; and overtreatment of questionable diagnosis.
For example, PSA, a marker elevated in prostate cancer, can also be increased in men with a
harmless enlargement of the prostate known as benign prostatic hypertrophy (BPH). In these
cases, the finding of an elevated PSA value can lead to unnecessary testing and potentially
harmful treatments, such as a prostate biopsy.
*Screening is most effective when it is conducted in populations at a high risk for
developing the disease, such as certain ethnic groups or those with a family history for a
particular type of cancer.
For example, alpha-fetoprotein (AFP), a tumor marker for hepatocellular carcinoma, is not used
for screening in the United States, but is used to screen people in China, where the incidence of
liver cancer is high.

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In high-risk populations, the predictive value of the tumor marker will be highest.
In other words, a positive test result is most likely to be found in a person who truly has the
disease, whereas a negative result is most likely to occur in a person who truly does not have the
disease.
Diagnosis Using Tumor Markers
 Second application of tumor markers; to help physicians distinguish between different

diseases with similar symptoms, a process called differential diagnosis
differential diagnosis - is a list of possible conditions or diseases that could be causing

your symptoms based off of this information
Ex if a computerized tomography (CT) scan revealed the presence of a lung nodule, histological
examination of a lung biopsy could help differentiate whether the nodule was caused by cancer
or an-other disease process, such as an infection. Follow-up staining of the biopsy for tumor
markers could help determine the neoplasm’s tissue origin.
Prognosis Using Tumor Markers
high concentration of a tumor marker at the time of diagnosis or increasing levels of

a tumor marker overtime can indicate the presence of an aggressive tumor
tumor markers can also be used to determine what type of therapy would be most
effective to patient
Examples:
in breast cancer anti-HER2 agents such as trastuzumab work best in patients agents such
as trastuzumab work best in patients whose tumors overexpress the HER2 protein or gene;
antiendocrine therapies such as tamoxifen are suitable for patients whose tumors
overexpress the estrogen receptor
Monitoring Patient Response to Treatment Using Tumor Markers
One of the most important uses of tumor markers

Can be done because the level of a serum tumor marker often correlates with the amount
of tumor in the patient
A significant decrease in the concentration of a tumor marker after surgery,
chemotherapy, or other treatment indicates that the therapy has been effective in
shrinking the tumor
In contrast, an increasing level of the marker after a return to normal indicates that the
tumor has recurred and that more aggressive treatment may be needed.

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The clinical significance of monitoring a tumor marker is illustrated in Figure 17–3. This figure
shows tumor marker levels from a hypothetical cancer patient who has been treated with
surgery and two chemotherapy drugs. As expected, the level of the tumor marker in the
patient’s serum declined after the initial tumor mass was removed by surgery. However, after a
few months, the concentration of the tumor marker began to increase, indicating that the tumor
had recurred. The tumor was unresponsive to Chemotherapy #1, as reflected by the sustained
elevation in the marker after treatment with that drug. This prompted a change in treatment to
Chemotherapy #2, which was successful in decreasing the amount of tumor present in the
patient, as indicated by the decline in the tumor marker to an undetectable level.
Serum Tumor Markers although there are scores of possible tumor markers in the literature,
only a few have FDA approval.
However, many tests for non-FDA-approved markers are available to clinicians, with a
notation on the laboratory report stating that results are for research use only. The National
Academy of Clinical Biochemistry (NACB) has published consensus guidelines regarding the
clinical use of tumor markers.
Alpha-Fetoprotein (AFP)
Alpha-fetoprotein (AFP) is a 70,000 MW glycoprotein that is similar to albumin in its physical

and chemical properties.It is classified as an oncofetal antigen because it is synthesized by the
fetal liver and yolk sac and is abundant in fetal serum, but declines to low levels (10–20 ug/L)
by 12 months of age. AFP is frequently elevated in patients with primary hepatocellular
carcinoma (HCC) and nonseminomatous testicular cancer, but can also be elevated in other
types of cancers and in nonmalignant conditions such as pregnancy and hepatitis

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AFP is the most widely used tumor marker for HCC, serving as a tool in diagnosis, staging,
prognosis, and monitoring patients undergoing therapy.
The sensitivity of AFP for HCC is 41% to 65% and its specificity ranges between 80% to
94%.25 As a result, the utility of AFP in screening for HCC has been a matter of debate.
However, screening for HCC with AFP is routinely performed in high prevalence areas of the
world such as China and Southeast Asia. In the United States, AFP screening is usually
conducted in patients with a high risk for HCC, along with liver ultrasound.
Studies have shown that the diagnostic utility of AFP may be improved by testing specifically
for the isoform AFP-L3, which has a stronger correlation with HCC, and by combining AFP
with other laboratory markers, such as DCP (des-γcarboxy-prothrombin), the liver enzyme ALT
(alanine aminotransferase), and platelet count.
In addition, high levels of AFP are associated with a poor prognosis in patients with HCC,
whereas decreasing levels over time indicate a good response to therapy.
AFP is also an established tumor marker for nonseminomatous germ cell cancers of the testes
(NSGCT).16,27 This marker, along with other markers such as human chorionic gonadotropin
(hCG; see section later in this chapter) and lactate dehydrogenase (LDH), plays an important
role in patient diagnosis, tumor staging, therapeutic monitoring, and detection of relapse. AFP is
elevated in 10% to 20% of patients with stage I NSGCT and in nearly all patients in later stages
of the disease.16 As in HCC, increased concentrations are associated with a poor prognosis,
whereas declining levels reflect responsiveness to therapy.
In addition to its applications as a tumor marker, AFP is widely used as a marker to detect
abnormalities in the fetus. An increased level of AFP in the serum or amniotic fluid of a
pregnant woman is seen with open neural tube defects such as spina bifida, whereas low levels
of AFP are associated with Down syndrome.
Cancer Antigen 125 (CA 125)
Cancer antigen 125 (CA 125) is a large, heavily glycosylated, mucin like protein that is a
marker for ovarian cancer. This marker is not unique to ovarian tumors because it is also found
in the normal ovary as well as other tissues, including the endocervix, endometrium, fallopian
tubes, pleura, pericardium, peritoneum, and epithelial tissues of the colon, pancreas, lung,
kidney, prostate, breast, stomach, and gallbladder.
CA 125 is considered the best marker for ovarian cancer. It has multiple applications to the
disease, ranging from screening and diagnosis, to prognosis and monitoring response to therapy.
Serum CA 125 levels greater than 35 kU/L are considered to be above normal. Although 90%
or more of women with ovarian cancer in stages II to IV have elevated CA 125, the marker is
not recommended for screening of the general population because it lacks sensitivity and
specificity. Elevated CA 125 levels are only seen in 50% to 60% of women with stage I ovarian
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therefore, generalized screening would miss about half of the women during the period when
the disease is most treatable. In addition, CA 125 is not specific because it can increase during
pregnancy menstruation, or as a result of benign gynecological conditions such as
endometriosis, non gynecological conditions involving inflammation, and other malignancies.
However, annual CA 125 testing, together with transvaginal ultrasound, is recommended for
women with a family history of ovarian cancer because early detection and intervention is likely
to be beneficial in this population.
The value of CA 125 can also be seen in clinical applications other than screening. For
example, an elevated CA 125 concentration combined with imaging has been shown to be
highly sensitive and specific for a differential diagnosis of ovarian cancer from benign pelvic
masses, especially in postmenopausal women.
Serial CA 125 testing is beneficial in monitoring a patient’s response to chemotherapy and is

recommended before treatment is initiated, during treatment, and during patient follow-up.
Rising concentrations of CA 125 over time can predict recurrence of the disease. Persistent
elevations of CA 125 or an initial CA 125 value greater than 65 kU/L point to a poor prognosis.
Carcinoembryonic Antigen (CEA)
Carcinoembryonic antigen (CEA) is a glycoprotein with a molecular weight of 180,000 to

200,000, depending on the exact structure of its carbohydrate side chains.
CEA was the first oncofetal antigen to be discovered. In 1965, Gold and Freedman described its
presence in tissues from the fetal colon and colon adenocarcinoma, as well as its absence in
colon tissues from healthy adults IIncreased serum concentrations of CEA are associated with
colorectal cancer because CEA is the most widely used marker for that cancer.
The main application of CEA is in monitoring patients undergoing therapy for colorectal
cancer.It is recommended that the medical team obtain a baseline CEA value from the
laboratory just before therapy, followed by CEA testing every 1 to 3 months during active
treatment.Increasing CEA levels are a highly sensitive indicator of liver metastasis and can
detect recurrent colorectal cancer by an average of 5 months before clinical symptoms
appear.CEA measurement should be used in conjunction with clinical examination, radiological
testing, and histological confirmation to maximize its sensitivity in detecting disease recurrence.
CEA levels can also be used in determining the most appropriate treatment for colorectal cancer
patients because those with higher baseline levels before surgery tend to have a poorer
prognosis.However, CEA is not recommended for colon cancer screening because of its low
sensitivity and specificity in this situation.
CEA is not increased in all patients with colorectal cancer and elevated CEA levels can be
present as a result of other conditions, including colitis, diverticulitis, irritable bowel syndrome,
and nonmalignant liver disease. Cigarette smoking can cause an increase in CEA level to nearly
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that of nonsmokers. CEA levels can also be elevated in other cancers, notably those of the
breast, gastrointestinal tract, pancreas, and lung.
Human Chorionic Gonadotropin (hCG)
Human chorionic gonadotropin (hCG) is best known as the “pregnancy hormone” because it is
synthesized by trophoblasts, cells that contribute to development of the placenta and promote
implantation of the embryo. Accordingly, it rises during the first few weeks of gestation, when
it can be detected in the blood and urine of pregnant women. In addition, hCG can be produced
by certain malignant tumors; elevations are associated with germ cell tumors of the ovary and
testes as well as choriocarcinoma, a rare type of cancer that is caused by malignant
transformation of the trophoblast cells.
In these tumors, testing for hCG is recommended as an aid to diagnosis, prognosis, monitoring
response to therapy, and detection of recurrence. hCG is a 45,000 MW glycoprotein that is
composed of an α subunit, which is shared by luteinizing hormone (LH), follicle-stimulating
hormone (FSH), and thyroid-stimulating hormone (TSH), and a β subunit that is unique to hCG.
Serological tests can measure either intact hCG or the hCG β subunit; the presence for both
should be tested to monitor patients with testicular cancer. This is because some patients may
only produce the
 subunit and detection of only the intact form can result in false-negative results.Because
hCG levels can also increase in men as the result of malfunction of the testes, it is important to
observe rising values in sequential tests before making a diagnosis of testicular cancer.
Elevations of hCG can also occur as a result of gonadal suppression caused by chemotherapy
and do not necessarily indicate tumor recurrence.
Prostate-specific antigen (PSA)
is the most widely used marker for prostate cancer. It is a 28,000 MW glycoprotein that is
produced specifically by epithelial cells in the prostate gland. PSA was first discovered in
semen, where its function is to regulate the viscosity of the seminal fluid to facilitate mobility of
the sperm cells.
Its presence was subsequently noted in serum, where it is frequently elevated in patients with
prostate cancer. The specificity of PSA for the prostate gland led to its routine use as a
screening test for prostate cancer. Since its approval by the FDA in 1994, PSA testing has
resulted in a dramatic increase in the detection rate of early-stage prostate cancer and in the rate
of 5-year patient survival.
Despite these successes, general screening for prostate cancer has been a controversial issue
because it may potentially lead to unnecessary testing and treatment. There are several reasons
for this concern. Although PSA is specific for prostate tissue, it is not specific for prostate
cancer. PSA can also be elevated in other conditions affecting the prostate gland, such as benign
prostatic hyperplasia (BPH), an enlargement of the prostate gland that commonly occurs as men
age, or prostatitis, an inflammation of the gland occurring as a result of infection or
irritation.Transient increases in PSA levels can also occur if samples are collected shortly after
ejaculation, digital rectal examination (DRE), or prostate manipulation. As such, there is
concern that general PSA screening can lead to the performance of unnecessary prostate
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In addition, many prostate cancers are slow growing and would be unlikely to cause death
during an older man’s remaining life span.
Therefore, some clinicians believe that it may be better to carefully monitor the condition over
time, by active surveillance and observation (“watchful waiting”), than to initiate treatment for
early-stage prostate cancer that could decrease quality of life.Large clinical trials that tested
thousands of men to determine the benefits and harms of PSA screening have produced
conflicting results. As a result, there is disagreement among major agencies about whether PSA
screening should be performed and how it should be implemented.
For example, the American Cancer Society recommends annual PSA screening in conjunction
with DRE for all men over the age of 50 and the American Urological Association recommends
that routine screening be conducted from ages 55 to 69 and age 70+ if life expectancy is greater
than 10 years, whereas the U.S. Preventative Services Task Force does not recommend
screening at all. Earlier and more frequent screening may be recommended for men at higher
risk for prostate cancer, notably those with a family history of the condition, those who have a
known or suspected related genetic mutation, or those of African American ancestry.
Prostate biopsy is recommended for men with a total PSA value greater than 4.0 ng/mL to
determine whether the elevation is caused by malignancy. Another application of PSA testing is
to assist in the diagnosis of prostate cancer. Great effort has been expended to distinguish
between BPH, weakly aggressive prostate cancers, and highly aggressive cancers using PSA.
Modifications in PSA testing may be helpful in making this differentiation. One modification
involves testing for free PSA and the naturally occurring PSA-α-1-antichymotrypsin complex,
in addition to total serum PSA.
This combination increases the specificity of testing because the proportion of free PSA is
higher in benign conditions, whereas the proportion of complexed PSA is greater in prostate
cancer. Because free PSA quickly degrades at temperatures above 4°C, it is important to
perform testing within 3 hours of sample collection or to store the sample at –70°C if a longer
time interval is required.
Another approach is to calculate the PSA velocity (PSAV), or the rate of increase in PSA values
over time. PSAV is calculated as the difference in PSA concentration divided by the number of
years spanning the interval between sequential tests (reported as ng/mL/year). The rationale for
this approach is that PSA will increase more rapidly if a growing tumor is present. A PSAV
greater than 0.75 ng/mL/year has been shown to be strongly associated with the presence of
prostate cancer.
To rule out the possibility that an increase in PSAV is because of an infection of the prostate
gland, a repeat measurement of PSAV can be conducted after a course of antibiotics is
administered. Another proposed strategy to increase the performance of PSA testing is to
calculate the PSA density (PSAD). The rationale behind this concept is that an increase in
serum PSA is more likely to be caused by the occurrence of cancer in a man with a small
prostate gland versus a large prostate gland.
PSAD is calculated as the ratio of total PSA to the prostate gland volume. Although this
approach appears to increase the specificity of PSA for prostate cancer, it requires performance
of transrectal ultrasonography, which can be time consuming, costly, and yield a less-than-
perfect measurement of the prostate gland volume.PSA testing also plays an important role in
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known to have prostate cancer. PSA values, in conjunction with histological observation of
prostate biopsy tissue, can be used to predict the stage of prostate cancer and to guide
physicians in determining optimal treatment. In addition, a rapid rise in PSAV or in the amount
of time it takes for the PSA level to double are indicators of more aggressive disease.
A persistently high level of PSA after radical prostatectomy indicates that residual disease is
present. When surgery is successful in removing the tumor, PSA will decrease to undetectable
levels. Rising PSA levels after surgery are a sign that the malignancy is recurring and can
precede other indicators of disease recurrence by many years. PSA testing is also a sensitive
indicator of disease recurrence in men who have undergone hormonal therapy, but is less
sensitive in detecting recurrence after radiation therapy because circulating PSA levels decline
more slowly after that type of treatment
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Laboratory Detection of Tumors

& Immune System and Tumors Interaction
LABORATORY DETECTION OF
TUMORS ● Three types of laboratory methods:
gross and microscopic morphology of tumors
immunohistochemistry or automated immunoassays
molecular diagnostics to detect genetic mutations in the malignant cells.
Tumor Morphology
 The processing of suspected tumor tissue with gross dissection and preparation of slides
for microscopic analysis.
 The following are applied to the slides to enhance visible feature:
tumor marker antibodies
special stains
nucleic acid probes
 Considerable skill is required to accurately diagnose cancer on the basis of morphology.
 The final diagnosis is generally made with supplemental clinical information and
additional testing.
Immunohistochemistry
 uses labeled antibodies to detect tumor antigens in formalin-fixed or frozen tissue sections
of tumor biopsy material
formalin-fixed sections - treated with heat before testing to make the antigen
epitopes accessible.
 indirect staining method > direct staining method
is used because larger immune complexes are formed, providing more sensitive
amplification of the signal.
 Immunofluorescence provides a greater dynamic range than immunochromatographic

staining
useful for the identification and quantification of co-localized proteins
 Binding is visualized by:
light microscopy - after the addition of the appropriate chromogen in the case of
enzyme labels
fluorescent microscopy - when fluorescent labels are used
● Use of positive and negative control tissues are essential for accurate results.
Negative controls - necessary to ensure that the staining observed is because of
antibody binding and not the background (i.e., nonspecific reactivity)

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Positive controls - confirm that the antibody reagents are working properly.
Normal tissue on the same slide can serve as an excellent internal control.
Accuracy of the results is also increased when a broad panel of antibodies is used.
Clinically, immunohistochemistry is used as an effective technique of classifying tumors

of uncertain origin because many tumors can have a similar appearance histologically.
Tumor marker must be differentially expressed in the tumor of origin as compared with
other tumors.
The first step in immunohistochemistry is to broadly classify the tumor into one of three
major lineages: epithelial, mesenchymal, or hematopoietic.
can be accomplished through the use of stains for specific markers
Cytokeratins - intermediate filaments found in all types of epithelial cells, are
used as markers for tumors of epithelial lineage.
Vimentin - an intermediate filament that is found in most mesenchymal cells, is
used as a marker to indicate the presence of a sarcoma or melanoma
specificity is low because it is expressed in some other tumor types
as well CD45 - a WBC marker
used to identify hematopoietic malignancies
Immunoassays for Circulating Tumor
Marker Immunoassay - a method that is easily detect specific
molecules.
Tumour markers - also called “biomarkers” are molecules that may be present in higher than
usual concentrations in the tissue, serum, urine, or other body fluids of patients with cancer.
Serum tumor markers - Serum tumour markers may aid cancer diagnosis, assess prognosis,
guide choice of treatment, monitor progress during and after treatment, and/or be used as
screening tests.
are most commonly measured by immunoassays because they are highly sensitive, lend
themselves to automation, and are relatively easy to use.
Note: Despite their advantages, immunoassays can be affected by several factorsthat need
to be considered when results are interpreted. These factors are related to the use of
antibodies as reagents.
Factors:
First - antibody reagents from different manufacturers can vary greatly in terms of what they
detect, particularly if monoclonal antibodies are used.
 Monoclonal antibodies are laboratory-made proteins that mimic the immune system's
ability to fight off harmful pathogens such as viruses.

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Note: It is important to use the same method for monitoring patients over time because results
can be affected if patients change clinics or laboratories.
Note: If laboratories switch methods, they must provide a transition period during which
samples are measured by both methods and specimens are archived until new data is established
for each patient.
Second - although antibodies are employed for their specificity, it is not absolute.
 Antibodies will cross-react with similar structures, which is particularly problematic when
the crossreacting substances are present in excessive amounts, as can occur with cancer.
Example: α subunit of Human chorionic gonadotropin (hCG) is virtually identical to the α

subunit of luteinizing hormone (LH), and the β subunits of the two hormones are 80%
homologous, so epitope choice is quite important.
(Human Chorionic Gonadotropin (hCG) - is a chemical created by trophoblast tissue, tissue
typically found in early embryos and which will eventually be part of the placenta. An
unchanged level of hCG or an increase in levels may mean that the cancer is not responding to
treatment, is still growing or has come back (recurred).)
Note: Tumors may produce free α and free β chains in addition to intact hCG, so an
immunochemical method that detects only β chain epitopes to minimize LH interference will
measure something completely different than a method that measures intact hCG.
A third factor that can affect immunoassay results is antigen excess.
 By virtue of their unchecked growth and aggressive metabolism, some neoplasms may
produce massive amounts of tumor marker molecules.
 The excess of tumor antigen as compared with reagent antibody can result in a postzone
effect.
Postzone - is a well-known limitation of immunoassays in which saturation of the antibodies

with antigen inhibits the cross-linkage required to visualize the reaction.
Note: When the measurements exceed the linear range of reportable results, this phenomenon is
called the high-dose hook effect, because of the shape of the curve that depicts the relationship
of the concentration of the analyte and the intensity of the reaction signal.
● This effect can result in a falsely decreased measurement in the area of antigen excess.

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 Therefore, the sample must be diluted to determine its value within the reportable range.
It is critical that criteria be developed to identify situations in which the hook effect may
be present so that specimens can be systematically diluted and accurate results obtained.
DILUTED - (of a liquid) weakened by the addition of water or another solvent.
A final problem with immunoassays is that interference can be caused by the presence of
endogenous heterophile, anti-animal, or autoantibodies in the patient sample.

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Autoantibodies
are produced in response to self-antigens, and/or antibodies that mistakenly target and
react with a person's own tissues or organs. One or more autoantibodies may be produced
by a person's immune system when it fails to distinguish between "self" and "non-self."
Heterophile antibodies
are capable of reacting with similar antigens from two or more unrelated
species. antibodies usually have low avidity but can react with a broad
range of antigens.
Anti-animal antibodies
Are species-specific, higher avidity antibodies that are produced by patients as a result of
passive immunotherapy with mouse monoclonal immunoglobulins or polyclonal
antibodies of animal origin
Note: These antibodies can affect

immunoassays in more than one way, causing
either falsely elevated (most commonly) or
falsely decreased test results.
The principle of interference resulting in a

false-positive result.
False positive = sa result merong sakit, pero wala naman palang sakit si patient.
False negative = sa result walang sakit, pero meron palang sakit si patient.
Example 1: a tragic case involving false-positive hCG results reported from an automated
analyser led to several women having unnecessary chemotherapy or hysterectomies for a cancer
that was suspected but not actually present.
Example 2: Pregnancy Test
 A falsely increased - results when there is an abnormal pregnancy and when there is a
biochemical pregnancy that means the Beta HCG has increase but no baby.
 A false decrease - has come negative but the px is already pregnant.
 falsely increased results by mechanisms similar to those shown in Figure 17–5,

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16. falsely decreases are also possible. In these situations, the patient antibody binds to the
marker of interest or blocks the binding of the labelled detector antibody, preventing the
formation of the immune complex “sandwich” and leading to a falsely low or negative
result.
To resolved:
 To confirm the presence of interfering antibodies, the sample can be diluted and the
linearity of the results can be analyzed.
 The laboratory can also test directly for the antibodies themselves
 The likelihood of interference by endogenous antibodies can be reduced by pre-treatment
of the sample with commercial blocking reagents.
(These reagents are typically mouse or rabbit immunoglobulins that bind to the interfering
antibodies and neutralize them).
Interference with tumor marker tests can also be caused by factors that can affect other
immunoassays, such as icterus, lipemia, and hemolysis.
Note: In any case, patient results should not be reported until the interference issue is resolved.
 Additional recommendations should be considered in the performance of tumor marker

tests.
 The presumption that a relatively low number of people being screened actually have
cancer and that it would be worse to miss a cancer than to do further testing on a normal
person to exclude cancer.
 Thus, a very high number of false positives is expected because of low disease prevalence.
 Establishment of a baseline level at initial diagnosis followed by serial testing over time
can provide valuable information when a patient is being monitored for response to
treatment or tumor recurrence.
Note: In this case, it is not a single absolute value of the tumor marker that is important, but
rather the upward or downward trend when the marker’s biological half-life is considered.
 When performing serial testing, each test should be performed by the same laboratory with
the same test kit to minimize variations in results. Testing for multiple markers, if
possible, will increase sensitivity and specificity.
 The limitations of immunoassays have prompted a search for more specific and sensitive
markers using molecular and proteomic technologies.
Molecular Methods in Cancer Diagnosis
 Because cancer is a disease process that involves many genetic alterations, scientists have
searched for changes in the genome that characterize the various types and subtypes of
cancer.

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 Identification of genetic mutations has become an important tool in cancer diagnosis and
determination of prognosis.
 Molecular diagnostics are a fundamental component of precision medicine, the approach
in which each person receives the best treatment for his or her particular health condition
based on an analysis of DNA, RNA, protein, or related molecules.
Genetic Biomarkers
● Molecular characterization is an essential component of testing for hematologic malignancies.
For example:
 The chromosome 9/22 [BCR/ABL] gene rearrangement is a well-established biomarker for

Chronic myelogenous leukemia CML.
 Rearrangements in immunoglobulin genes and T-cell receptor (TCR) genes are used to
identify malignant clones of B cells or T cells, respectively, in leukemias and lymphomas.
 Genetic analysis is also helpful in the diagnosis of solid tumors.
For example:
 Mutations in the MSH genes of the DNA mismatch repair system are helpful in the
diagnosis of hereditary colorectal cancer; these alterations create microsatellite instability,
an alteration in the length of repetitive DNA sequences that can be visualized by
molecular testing.
 Molecular analysis of gene expression for the estrogen receptor (ER), progesterone
receptor (PR), and HER2 can create a genetic profile that is used to classify breast cancer
patients into subtypes that provide prognostic information and guide physicians in
choosing therapeutic plans that have the best chances of success.
Genetic biomarkers can also be used for:
Prospective and post-diagnostic evaluation of malignancies.
Prospective markers:
Can provide valuable information regarding the risk for an asymptomatic person to develop a
particular type of cancer, the growth rate of the cancer, or the development of metastatic
disease. For example, women with hereditary mutations in the BRCA1 or BRCA2 genes carry a
40% to 80% lifetime risk for developing breast cancer, as well as an 11% to 40% lifetime risk
for ovarian cancer.
Post diagnostic genetic markers:
Are used to guide clinicians in making appropriate treatment decisions for known cancer
patients.
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 By mid-2014, the FDA had approved over 40 targeted cancer drugs for administration in
cancer patients with the appropriate genetic biomarker. The list of approved therapies
Includes drugs that target tumors with alterations in the:
 BRAF gene (metastatic melanoma),

 KRAS gene (colorectal cancer),
 EGFR gene (non-small cell lung cancer; NSCLC),
 ALK gene (NSCLC),
 HER2 gene (breast cancer),
 ESR1, and
 PGR genes (breast cancer)
Nice to know: Testing for these and other genetic markers is incorporated into the clinical
practice guidelines published by the National Comprehensive Cancer Network and the College
of American Pathologists.
Testing for biomarkers has been made possible through scientific advances in molecular
techniques, including:
 Nucleic acid amplification techniques (NAAT),
 fluorescent in situ hybridization (FISH),
 microarray, and
 DNA sequencing.
In NAAT, such as:
 Polymerase Chain Reaction (PCR)
Millions of identical copies of a specific target sequence within a nucleic acid are
synthesized in the laboratory from an original DNA template derived from the cancer cell
population.
These methods are used to amplify the sequence that potentially contains the genetic
mutation of interest, allowing tiny changes in the sequence to be detected by the
differences in fragment sizes that can be visualized by gel electrophoresis.
Cytogenetics
Cytogenetics- studies play a large role in the diagnosis and management of cancer.
Karyotype analysis- has been used for many years to detect the chromosomal
abnormalities associated with many cancers. The number of these aberrations can
increase as the disease advances.
Abnormality that can be detected by karyotyping:

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Aneuploidy - a condition in which individual chromosomes are gained or lost.

Deletion - in which a portion of a chromosome has been lost.
Rearrangement - involving breakage of two different chromosomes and translocation
of the fragments onto the opposite chromosomes
 The development of FISH has allowed chromosomal abnormalities to be characterized

more precisely at a molecular level.
In FISH:
 Interphase cells from the patient’s tumor are incubated with fluorescent-labeled nucleic
acid probes that are complementary to the sequence of interest.
 Cells containing the sequence will bind the probes and can be visualized with a
fluorescent microscope.
In oncology:
FISH is most often used to detect chromosome rearrangements and gene amplification.
To detect a chromosome translocation, such as the one seen in the BCR/ABL rearrangement
characteristic of CML, two single probes are used, each specific for one of the two
chromosomes and each labeled with a different fluorochrome (for example, red and green)
Normally, each cell should have two red signals and two green signals.
If a translocation has occurred,
a fusion probe signal is
generated, in which the
red signal is adjacent to
the green signal,
producing a yellow color
(see Figure 18–11B).
To detect gene amplification,

the probe hybridizes to the gene of interest.
If that gene is over expressed, multiple copies of the fluorescent signal representing the
probe will be observed.
This application of FISH is commonly used to detect the HER2 gene amplification seen in
some cases of breast cancer.
Note: FISH is a highly specific method to detect molecular abnormalities in tumor cells, it can
only detect gene sequences that are complementary to the probes used; as such, it may not
detect rare, tiny deletions.
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Microarrays
Microarray - technology has been developed to test for panels of markers, rather than
individual mutations.
In this method:
 single-stranded DNA or RNA from the tumor is tagged with a fluorescent label and
incubated with known nucleic acid sequences that have been spotted onto different areas
of a membrane.
 The sample will hybridize to any complementary sequences on the tiny spots, allowing
for simultaneous testing of the specimen for multiple genes.
Microarrays can also be used to compare the

levels
of gene expression in cancer cells with
those of
normal cells by using two different colors of
fluorescence to tag nucleic acid from each cell
type.
Note 1: The microarray is interpreted with

a
fluorescent reader and analysis software.
Note 2: Microarrays can screen tens of thousands of gene sequences at the same time for their
ability to bind to the sample nucleic acid, thus generating an enormous volume of information.
Note 3: Sophisticated mathematics and computer software are necessary to analyze the vast
amount of biological data.
Bioinformatics - collection and analysis of such data.
It is hoped that this approach will help uncover molecular signatures that can
characterize specific tumor types and subtypes to provide better information
regarding patient diagnosis and prognosis.
Gene expression arrays, such as the MammaPrint and Oncotype Dx for breast
cancer, are being commercially developed to aid in predicting the likelihood of
cancer recurrence and guiding treatment decisions in cancer patient.
Next Generation Sequencing
provides large amount of data
thousands of genes within the tumor can be sequenced simultaneously in just a few hours
to identify genetic variations
detect metastases by analyzing DNA from tumor cells circulating in the peripheral blood
plays a major role in generating an enormous volume of data for The Cancer Genome
Atlas (TCGA).

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TCGA is conducting a large-scale collaborative project funded by the National

Cancer Institute and the National Human Genome Research Institute to catalogue
and characterize the genomic changes that occur in cancer.
The goal of the project is to identify all of the pertinent genomic alterations in
several tumor types.
Proteomics
 Analysis of the entire protein complement of a cell population
 The analysis is being done through the use of two-dimensional electrophoresis coupled
with tandem mass spectrometry (MS/MS), surface-enhanced laser
desorption/ionization mass spectrometry (SELDI-TOF), or more recently, antibody
arrays.
 Antibody arrays - analysis that is being done recently
Advantage: do not require fractionation or depletion of high abundance proteins to
detect proteins that are present in lower concentrations
 Biomarker profiling - allow laboratories to identify unique patterns of proteins and their
metabolites that are characteristic of particular types of cancer
 The data generated from proteomic methods may help clinicians diagnose cancer earlier
and lead to the development of personalized therapies that can effectively target the
underlying biology of this highly complex disease entity.
IMMUNE SYSTEM AND TUMORS INTERACTION

Paul Ehrlich (100 years ago) - immune system plays an important role in eliminating
tumor cells.
Immunosurveillance
Proposed by F. MacFarlane Burnet and Lewis Thomas in 1950’s
States that the immune system continually patrols the body for the presence of cancerous
or precancerous cells and eliminates them before they become clinically evident.
The protective role of the immune system against tumors has also been supported by
clinical evidence in humans.
Higher incidence of cancer - observed in transplant patients who received
immunosuppressive therapy and patients with immunodeficiency diseases than in the
general population
Cancer rises after the age of 60
Immune Defenses Against Tumor Cells

 Main components of the immune system against tumor cells - NK cells, macrophages,
cytotoxic T cells (CTLs), cytokines, and possibly T helper (Th) cells and antibodies.

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Innate Immune Responses

 Mediated by NK cells and macrophages
 Natural Killer cells
act in a mechanism that is similar to CTLs, but can kill tumor cells without prior
sensitization to tumor antigens
activated to kill cells that lack class I MHC molecules
Activating receptors on NK cells ➡ bind to tumor antigens ➡ initiating
intracellular signals (degranulation and the release of perforin and granzyme) ➡
apoptosis.
most effective against malignant cells
Activity is increased by incubation with IL-2.
In vitro culture of peripheral blood cells or tumor infiltrating lymphocytes (TILs)
with IL-2 results in the generation of lymphokine-activated killer (LAK) cells
which destroy tumor cells by the same mechanism as NK cells but are much more
potent.
 Macrophages
activated in vitro by IFNγ to possess tumoricidal capabilities
kill tumor cells by the same mechanisms they use to kill infectious organisms,
including release of lysosomal enzymes, reactive oxygen species, and nitric oxide.
produce TNF-α which cause necrosis of tumors by inducing local inflammation
and thrombosis in the blood vessels within the cancerous mass

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Adaptive Immune Response

 Mediated by CTLs, antibodies, activated Th cells and cytokines
 cytotoxic T lymphocytes (CTLs)
initiated by dendritic cells by processing tumor antigens and displaying the
peptides derived from these antigens on their surface in conjunction with class I
MHC molecules Cross-presentation - exogenous tumor antigens are presented in
context with
class I MHC molecules rather than class II MHC molecules
 The APCs present the tumor peptide-antigen complexes to specific TCRs on the surface of
the CTLs and provide costimulatory signals that promote maturation of the CTLs
 Mature CTLs - use their antigen-specific TCRs to bind class I MHC on the surface of the
tumor cell.
 granules migrate toward the plasma membrane and release cytotoxic proteins within the
synapse formed between the CTL and the tumor cell.
Perforin - creates pores in the membrane of the tumor cell
Granzyme - enter through the pores and cause apoptosis of the tumor target.
 NKT cells - express surface antigens of both NK cells and T cells,
are able to destroy tumor cells in a mechanism that is similar to the CTLs, but they
have a unique type of TCR that recognizes glycolipid antigens instead of peptide
antigens.
CD4+ Th cells - thought to be activated by dendritic cells through presentation of tumor
antigens in conjunction with class II MHC molecules.
IL-2 - can promote CTL development and enhance NK cell activity
IFNγ - activates macrophages and increases class I MHC expression on the tumor
cell surface.

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Immunoediting and Tumor Escape
 As the field of tumor immunology advances, scientists are recognizing that

immunosurveillance is only part of a broader process that explains the complex
relationship between the immune system and cancer. This process, termed
immunoediting, is thought to consist of three phases: elimination, equilibrium, and
escape (Fig. 17–7).
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Elimination
 Elimination phase of the cancer immunoediting process is essentially the same

as the immunosurveillance concept that was just discussed.
 If the immunologic mechanisms involved in immunosurveillance are highly effective,
they will likely result in complete elimination of the tumor.
 If the immune responses are not completely effective, some of the tumor cells will
remain in the body. The immunoediting hypothesis suggests that these cells will then
enter the equilibrium phase.
Equilibrium

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 In this phase, tumor cells are thought to enter a state of dynamic equilibrium with the
immune system, which keeps the altered cells under control so that they are not clinically
evident. During this period, tumor cells may remain dormant or evolve slowly over time.
 The dynamic interactions between the tumor and the immune system are thought to
shape the phenotype of the tumor and its ultimate outcome, hence the term
immunoediting.
As a result, the tumor may eventually be eliminated by the body,
establish permanent residence in the equilibrium phase, or evolve into a
phenotype that can escape the immune system and cause disease.
 During this phase, mutations can occur in the genetically unstable transformed cells.
Under selective pressure from immunologic forces of attack by cells in the tumor
microenvironment, some of the tumor cells may develop into genetic variants that are
resistant to immune defenses. These cells move past the equilibrium phase and enter the
escape phase.
Escape
 During this phase, the balance between immunologic control and tumor development is
tipped in favor of the neoplasm and tumor growth progresses, even in the presence of
anti-tumor immune responses.
 Cancer is a heterogeneous disease and tumors have developed a variety of strategies for
evading the immune system (see Fig. 17-7).
 Some of the escape mechanisms employed by tumors are a result of changes in the
edited tumors themselves, which lead to reduced immunogenicity.
EXAMPLE: some tumors downregulate the expression of tumor antigens or
MHC molecules on the cell surface, making them less likely to be recognized
by T cells.
 Other modifications may involve defects in components of the antigen processing
machinery associated with class I MHC molecules.
 Tumor antigens may also be masked by glycoproteins and glycolipids on the cell
surface, making them inaccessible to the immune system.
 Other alterations can result in tumor resistance to immune defenses.
EXAMPLE: impaired cell surface binding to perforin or defective apoptosis-
inducing receptors such as Fas have been noted in some tumors
 Another way that tumors can escape the immune system is to suppress anti-tumor
immune responses.
Tumors can do this directly by secreting immunosuppressive substances or
indirectly by recruiting T regulatory (Treg) cells, myeloid-derived suppressor
cells, or macrophages that produce cytokines such as transforming growth factor-β
and IL-10, which can inhibit protective immune responses.
 Another factor that may contribute to tumor progression is inflammation.
Although acute inflammation may be protective to the host, chronic inflammation
is believed to modify the cellular microenvironment in ways that promote the
development of tumors.

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Chronic inflammation and its associated proinflammatory cytokines may

contribute to tumorigenesis in a number of ways, including generation of
cellular stress and free radicals, production of growth factors that induce cell
proliferation, enhancement of angiogenesis and tissue invasion, and suppression
of adaptive immune responses.
IMMUNOTERAPHY
 The type of therapy used for a particular patient depends on the type of tumor present
and the stage of disease.
 Traditional therapies:
- surgery – to remove solid tumors

- radiation therapy - to reduce tumor size
- chemotherapy
- Hormone therapy - to target residual tumor cells and metastases.
 Also known as biological therapy or biological response modifier therapy

The GOAL: is to harness the ability of the immune system to destroy tumor cells.
 Immunotherapeutic
methods
major
types:
Active immunotherapy - patients are treated in a manner that
stimulates them to mount an immune response against their
tumors
Passive immunotherapy- involves administration of tumor-
specific antibodies or cytokines to patients who may not be able
to develop an adequate immune response
Adoptive immunotherapy – cells from the immune system are
provided to patients.
Active Immunotherapy and Cancer Vaccines
 In 1891, the bone sarcoma surgeon, William Coley, began the first systematic
study of immunotherapy.
He noted that cancer patients who developed an infection after surgery
experienced tumor regression and had a better prognosis than patients who
did not acquire an infection.
Inspired by this knowledge, he decided to inject one of his cancer patients with
Streptococcus pyogenes bacteria. To his amazement, the patient’s tumor shrank
and the patient became cancer free.

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He went on to treat additional patients and observed shrinkage of their

malignant tumors, although some of the patients died from infection because
the bacteria were alive and virulent.
Coley then developed a less dangerous version of his treatment, which consisted
of a mixture of killed S pyogenes and killed Serratia marcescens bacteria. This
formulation later became known as “Coley’s toxins” and was widely used by
Dr. Coley and other physicians for the next 30 years to treat patients within
operable bone and soft-tissue sarcomas.
However, Coley’s treatment was controversial and many doctors who used the
toxins did not get good results, particularly with other types of cancer. In 1962,
the FDA refused to recognize the formulation as an effective cancer therapy and it
became illegal to use Coley’s toxins to treat cancer.
Coley is considered as the “Father of Immunotherapy.”
Other agents have been used to nonspecifically boost the immune system.
Bacillus Calmette Guerin (BCG) - a live but weakened strain of
Mycobacterium bovis bacteria, is considered to be the treatment of
choice for noninvasive bladder cancer
 The development of prophylactic cancer vaccines offers an effective approach to

active immunotherapy.
These vaccines have been generated for the purpose of preventing virus-
associated cancers.
The human papilloma virus (HPV) vaccine is effective in preventing cervical
cancer and the hepatitis B virus (HBV) vaccine has been successful in reducing
the incidence of HBV infection of the liver and its associated complications,
including hepatocellular carcinoma.
These vaccines are designed to induce an immune response against tumor
antigens with the hope of eliminating existing tumor cells and producing long-
lasting immunity.
Early cancer vaccines used tumor cell lysates or whole tumor cells that were
inactivated by treatments such as irradiation.
Unfortunately, clinical trials found that most killed tumor cell vaccines did not
have a significant effect on patient survival; as a result, this approach is
generally not used today
 In one approach to developing these vaccines, the genes that code for TSAs are
identified and cloned in recombinant vectors such as viruses or bacterial plasmids.

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The vectors can be genetically engineered so that they produce peptides

that are recognized by CTLs, which, as we discussed, are important
effectors of tumor immunity.
Another approach is to use synthetic peptides or full length tumor proteins
in the presence of a vaccine adjuvant.
Examples of such vaccines:
HER2 antigen - to treat a subset of breast cancer patients
tumor immunoglobulin idiotype - to target B-cell lymphoma
MAGE-3 antigen - for some patients with melanoma or NSCLC
 An interesting strategy that has gained much attention is the use of dendritic cells
- To immunize patients against their own tumors.
- dendritic cells (DCs) are isolated from the cancer patient and incubated
with the pertinent tumor antigen or transfected with the gene that codes
for the antigen.
- The antigen-loaded DCs are then readministered to the patient, where they are
believed to function as potent APCs.
- Sipuleucel-T (Proveng)
- the only FDA-approved cancer vaccine at the time of this writing, is based
on this technology.
- The vaccine, which is designed to treat patients with advanced prostate cancer, is
produced by incubating the patient’s own peripheral blood cells with a fusion protein
composed of the antigen, prostatic acid phosphatase (PAP), and the cytokine GM-
CSF, which is thought to promote DC activation and induce a PAP-specific T-cell
response
 As previously mentioned, tumor cells can evade the immune response by

creating an immunosuppressive microenvironment
- T cells are unable to fully exert their tumoricidal potential.
- It may therefore be necessary to return the tumor environment into
an immunosupportive tissue before a cancer vaccine can be fully
effective.
- Scientists are attempting to do this by promoting local production of certain
cytokines (e.g., IL-12 and IFN-α) and using antibodies to eliminate Treg cells or
block molecules that inhibit immune responses
 Unlike vaccines for infectious diseases, which are used to prevent infection, most
cancer vaccines are immunotherapeutic, being administered after the disease has
occurred.
- They are frequently given to patients in the advanced stages of disease when
other treatment options have been exhausted.
- In this situation, the patient’s immune system has often been compromised
because of the disease process or the effects of chemotherapy; therefore,
response to the vaccine may be suboptimal.

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 In these cases, it may be more beneficial to provide the patient with components
of the immune system through passive or adoptive immunotherapy to more
effectively target destruction of the tumor.
Passive Immunotherapy
 Involves the administration of soluble components of the immune system to boost the
immune response.
 Two approaches to passive immunotherapy in cancer patients involve the administration
of cytokines to nonspecifically enhance the immune response and treatment with
monoclonal antibodies to target specific tumor antigens.
Cytokines
 Cytokines are small proteins that play an important role in regulating immune responses
by serving as chemical messengers that affect the interactions between cells of the immune
system.
 Two main applications of cytokines in cancer treatment:
Use as hematopoietic growth factors
Use as therapeutic agents
 Chemotherapy drugs inhibit cell division, they often adversely affect the development of
hematopoietic stem cells in the bone marrow, resulting in decreased production of WBCs,
red blood cells (RBCs), and platelets.
 Hematopoietic growth factors, also known as colony stimulating factors, can be
administered to patients to help them recover from or prevent these toxicities. Some of the
main colony stimulating factors that have been used to treat cancer patients are:
granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony
stimulating factor (GM-CSF), erythropoietin, and interleukin 11 (IL-11). G-CSF
stimulates hematopoietic stem cells to develop into granulocytes, whereas GM-CSF
stimulates hematopoietic stem cells to develop into granulocytes and monocytes, thus
reducing the patient’s risk for severe infections.
 Erythropoietin stimulates production of RBCs from the bone marrow and can be used to
treat patients with severe anemia.
 IL-11 stimulates the maturation of megakaryocytes, helping patients to recover from
chemotherapy-induced thrombocytopenia.
 The therapeutic application of cytokines is aimed at enhancing patients’ immune responses
to their tumors. Preclinical and clinical investigations have been conducted for the
interferons (IFNs), tumor necrosis factors (TNFs), and several interleukins.
 Two examples of cytokines have been widely studied are:
IFN-a
IL-2
 Interferons were the first cytokines that were used as biological response modifiers. IFN-
a has been the most commonly used IFN in cancer therapy and has been approved by the
FDA for the treatment of several types of cancer, including malignant melanoma, hairy
cell leukemia,

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chronic myeloid leukemia, and Kaposi’s sarcoma. IFN-a is thought to promote anti-tumor
effects by increasing tumor immunogenicity, enhancing dendritic cell responses to the
tumor, enhancing Th1 responses and cell-mediated cytotoxicity, promoting tumor
apoptosis, and inhibiting angiogenesis. Although high doses of IFN-a are associated with
better clinical responses than low doses of the cytokine, they also generate strong adverse
effects, including fever, asthenia (loss of muscle strength), neutropenia, and nausea and
vomiting.
 Of all the interleukins, interleukin-2 (IL-2) has been the most extensively studied. IL-2
induces T-cell proliferation and enhancement of CTL and NK cell function. However, clinical
trials revealed that systemic administration of IL-2 as immunotherapy was limited because of
its short half-life (fewer than 10 minutes) and serious adverse effects, including vascular
leakage syndrome, marked fluid retention, and shock. Although this cytokine is still used to
treat metastatic melanoma and advanced renal cancer, it is rapidly cleared from the body and
its most effective use may be to activate immunocompetent cells in vitro for adoptive
immunotherapy.
 Cytokines continue to be incorporated in immunotherapy, their use has been limited
because of the serious and sometimes life-threatening side effects associated with high-
dose systemic treatment.
 The cytokine network is very complicated and administration of a cytokine can have
multiple, and sometimes unwanted, effects.
For example, in addition to its immunostimulatory effects, IL-2 is also
thought to be necessary for the generation and maintenance of Treg cells,
which can be involved in enhancing tumor growth.
Monoclonal Antibodies
These antibodies are derived from a single clone of cells, providing for an abundant source
of highly specific antibodies directed toward one particular epitope of an antigen.
Monoclonal antibodies in cancer immunotherapy have been directed against 7 major
categories of antigens:
CD antigens
Glycoproteins
Glycolipids
Carbohydrates
Vascular targets
Stromal and extracellular antigens
Growth factors
These antibodies have different mechanisms of action, depending on their target. The
major approaches to monoclonal antibody therapy (Table 17-5).

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 Some monoclonal antibodies are directed against antigens found on the surface of the
tumor cells. These antibodies are believed to destroy the tumor through the same
mechanisms that are used to attack infectious organisms, namely opsonization,
complement-mediated cytotoxicity, and ADCC.
 A second group of immunotherapeutic monoclonal antibodies target surface receptors
involved in intracellular pathways that lead to the growth and immortality of cancer cells.
These receptors are expressed at higher-than-normal levels on epithelial cancers of the
colon, breast, lung, head, and neck. Therapeutic antibodies bind to these receptors and
block cell signals that are necessary for activation of molecular pathways involved in cell
growth and survival.
 A third group of monoclonal antibodies target antigens involved in angiogenesis or the
formation of blood vessels that are necessary to provide the oxygen and nutrients needed
for tumor growth. Many of the antibodies in this category are directed against the vascular
endothelial growth factor (VEGF) family of proteins or their receptors.
 A fourth group of monoclonal antibodies boost the immune response to the tumor by
blocking inhibitory pathways that inactivate T cells. This approach uses monoclonal
antibodies to inhibitory receptors such as cytotoxic T-lymphocyte antigen 4 (CTLA-4),
which prevents T-cell activation when bound to the CD80 (B7-1) or CD86 (B7-2) proteins
on APCs, and PD-1, a receptor on T cells that inhibits T-cell proliferation when it is bound
to programmed death ligand 1 (PD-L1).
 One strategy to increase the effectiveness of monoclonal antibodies involves linking them to
potent cytotoxic drugs that can be taken up the tumor cells. These products are known as
antibody-drug conjugates or immunotoxins. They reduce the systemic side effects of the
toxins by localizing a small number of toxic molecules directly to the tumor cells using a
tumor-

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specific antibody. After binding to an antigen on the tumor surface, the conjugate is
quickly internalized by the cancer cell through receptor -mediated endocytosis and
transported to the lysosomes. The cytotoxic drug is released from its antibody into the
cytoplasm of the tumor cell, where it exerts potent toxic effects.
1st immunotoxins, derived from the plant toxin ricin, or toxins from diphtheria-causing or
Pseudomonas bacteria, had some effectiveness against tumors, but produced toxic side
effects. Newer generation antibody-drug conjugates are made from modified toxins that
can be genetically engineered by cloning genes for antibody fragments with genes for the
adapted toxin.
Two FDA-approved preparations that have shown promise are:
Brentuximab vedotin - an immunotoxin directed against the CD30 antigen that is
used to treat Hodgkin lymphoma (HL) and systemic anaplastic large cell
lymphoma (sALCL)
Trastuzumab-DM1 - specificity for HER2 antigen and is used to treat patients

with HER2-positive metastatic breast cancer.
Monoclonal antibodies have been used to treat almost all major subtypes of cancer; As a
result, this treatment modality has been established as one of the most successful
therapeutic strategies for cancer in the last 20 years.
Monoclonal antibody treatment can cause toxicity if the target antigen is expressed on the
normal cells. Therapy with monoclonal antibodies may be ineffective if the antibodies are
unable to permeate through tumor tissues or bind their target antigen molecules with high
affinity.
Cancer cells can develop resistance to monoclonal antibodies, analogous to the
way that bacteria can develop resistance to antibiotics.
Adoptive Immunotherapy
Early experiments conducted in mice in the 1960s showed that lymphoid cells from mice
immunized with certain tumors were able to protect against tumor growth when they were
transplanted into genetically identical mice; this response was enhanced in the presence of
IL-2.
In the late 1980s, Dr. Steven Rosenberg and his colleagues, it was discovered that adoptive
immunotherapy could be applied to the treatment of human cancer. These scientists
isolated lymphocytes from surgically removed tumors of patients with metastatic
melanoma and grew them in the laboratory in the presence of IL-2. They found that these
cells, referred to as tumor-infiltrating lymphocytes (TILs), demonstrated potent
cytolytic activity against autologous melanoma cells.
Subsequent modifications of technique resulted in significantly improved patient
outcomes. Instead of administering the entire population of TILs, cells are subcultured and
individually tested for their reactivity to the tumor (Fig. 17-8).

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Alternative treatments being investigated involve the use of genetically engineered T cells.
One method to construct these genetically engineered cells involves isolating T cells from
patients with good anti-tumor responses and cloning the genes for their TCRs into viral
vectors that can be used to infect T cells from the patient to be treated. A second approach
involves isolating TCR genes from humanized mice that have been immunized with the
tumor antigen of interest and cloning these into recombinant vectors to deliver the
sequences to T cells from the cancer patient. A third method is to generate chimeric
antigen receptors (CAR). CARs are most often constructed by combining the antigen-
binding variable fragment of a monoclonal antibody to a tumor antigen with intracellular
domains of the TCR that provide activating signals to the T cells. CARs can target tumor
antigens in an MHC-independent manner.
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IMMUNOPROLIFERATIVE DISEASES
M ALIGNANT TRANSFORMATION OF HEMATOLOGIC MALIGNANCIES
CELL PROPERTIES
 Hematologic Malignancies are characterized by excessive accumulation of cells in the blood,
bone marrow, or other lymphoid organs.
 The accumulation of the malignancies is due to:
 Rapid proliferation of the cells
 Failure to undergo apoptosis
 Lymphoid malignancies are classified into two: leukemias and lymphoma
 Malignant cell of Leukemias are primarily present in the bone marrow and peripheral blood
 Malignant cells of Lymphoma arise in the lymphoid tissues (tonsils, spleen and lymph node)
 In addition to a failure of growth regulation, mutations can result in arrested maturation of a cell
→ some malignant hematopoietic cells may not develop into properly functioning mature cells
 Malignant and premalignant proliferation of cells can occur at any stage in the differentiation of the
lymphoid lineages.
 Cells of the immune system are at great risk for malignant transformation because the features that
characterize the development of malignancy are also a normal part of the immune response.
 Proliferation of T and B lymphocytes is an integral part of the immune response to an antigenic
stimulus
 High rate of mutations during gene rearrangement and affinity maturation → considered as a
normal part of lymphocyte maturation.
 Despite being affected by abnormal regulation, malignant lymphoid cells generally retain some or all
of morphological and functional characteristics of their normal counterpart
 Their characteristics cell surface antigens or secretion of immunoglobulin →it is often used to
classify lymphoid malignancies
 Dysregulative theory of lymphoma was developed and it was based largely on the animal experiments
 The concept of the theory was that lymphomas arise when persistent immunostimulation coincides
with an immune deficiency.
 Immune deficiency plays 2 important roles:
 First, the presence of an ineffective immune response can permit persistent stimulation by
failing to clear an infection.
 Second, the immune system is responsible for surveillance against malignancy.
 It is reported that patients with an immunodeficiency have a higher rate of malignancy especially
malignancies linked to a viral etiology, than individuals with a normally functioning immune system.
 The immune system is naturally diverse and heterogenous in its response against a wide range of
potential pathogens.
 Normal immune response → polyclonal
 Polyclonal are cells with different features such as antigen specificity all proliferate in response to an
immune stimulus.
 Malignancies are thought to arise from excessive proliferation of a single mutant parent cell to form a
clone of genetically identical cells
 Malignancy can often be diagnosed when a population of cells is found to be more uniform than normal
 Plasma cells produces only one type of immunoglobulins, the persistent presence of a large
amount of a single idiotype suggests malignancy.
 An increase in the amount of total immunoglobulin, without an increase in any specific
class is the characteristics of benign.
GENETIC CHANGES
 Malignancies are generally multifactorial in origin
 Malignant transformation is thought to be a multistep process involving exposure to
environmental agents
 The key genes involved in the malignant transformation
 proto-oncogenes -genes involved in a normal cell growth and division
 tumor suppressor genes -genes that control cell division by regulating the progression
of cells through the cell cycle and maintaining genetic stability of the cells by repairing
damaged DNA
 oncogenes -alteration of proto-oncogene which are involved in malignant transformation.
 The genetic alterations in malignant cells of hematopoietic origin include:
 Point mutations involving a change in a single nucleotide base
 Duplications or deletions of specific genes
 Chromosome translocations
 Hematologic malignancies are characterized by translocations involving the proto-oncogene c-
MYC.
 c-MYC
 It plays an important role in regulating cell growth
 It stimulates the transcription of several other genes involved in cell proliferation
 Overexpression of this gene can occur as a result of a rearrangement in which c-MYC is
placed under the control of a different gene promoter sequence.
 Translocation involving the c-MYC gene on chromosome 8 and
immunoglobulin m gene on chromosome 14 [t(8;14)]
 Burkitt’s lymphoma- a B cell malignancy associated with Epstein-Barr
virus (EBV) infection.
 As a result of persistent c-MYC expression, several genes that are involved in cell
proliferation are activated beyond normal levels.
 The high levels of c-MYC protein drive the affected cells to continually proliferate.
 Other Hematologic malignancies associated with genes that affect apoptosis:
 Follicular lymphoma have a [t(14:18)] gene translocation → portions of chromosome
14 (which contains the Ig heavy- chain genes) and chromosome 18 (which contains
an anti-apoptotic gene called BCL-2) are exchanged.
 BCL-2
 It induces production of an inner mitochondrial membrane protein that blocks apoptosis
 Therefore, the cells affected by this translocation do not die normally
 Even though the altered cells do not proliferate at an increased rate, an excessive number of
cells accumulate because their survival is enhanced compared with normal cells.
 Other characteristic translocations result in the production of a novel fusion protein:
 Chronic myelogenous leukemia → characterized by a translocation between the
BCR (breakage cluster region) on chromosome 9 and the c-ABL proto-oncogene
on chromosome 22.
 This results in a BCR/ABL fusion protein, which codes for a continuously activated
tyrosine kinase enzyme, causing unregulated cell division.
 Gleevec → “anticancer drug”
→ developed by the scientists which slows cell growth by inhibiting the activity
of the altered kinase.
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1
IMMUNOLOGY AND SEROLOGY MIDTERMS
HYPERSENSITIVITY
INTRODUCTION TO HYPERSENSITIVITY
Hypersensitivity
● Defined as Heightened state of immune responsiveness
● It is an exaggerated response to a typically harmless antigen that results in injury to the tissue, disease, or even death
Immunization (or Sensitization)

● To describe an immunologic reaction dependent on the host’s response to a subsequent exposure of antigen
● Small quantities of the antigen may favor sensitization by restricting the quantity of antibody formed.
● An unusual reaction, such as an allergic or hypersensitive reaction that follows a second exposure to the antigen, reveals the
existence of the sensitization.
Allergy
● Our basic understanding of allergy has evolved from the discovery of IgE which is the type of Immunoglobulin responsible for
allergic reaction
● The most significant property of IgE antibodies is that they can be specific for hundreds of different allergens.
● Allergens are antigens that trigger allergic reaction
● Example of allergens are: Animal dander, Pollens, Foods, Molds, Dust, Metals, Drugs, Insect stings
Allergy and Atopy

● Allergy originally meant any altered reaction to external substances
● Atopy refers to immediate hypersensitivity mediated by IgE antibodies
NOTE: Atopy only represents IgE mediated allergic reactions. While allergy, it also encompasses not only IgE mediated allergic
reactions but also certain reactions mediated by other types of antibodies, such as agglutinins (reactions of Type II), precipitins
(caused Type III reactions), and certain reactions of Type IV, mediated by lymphocytes and not antibodies (example: contact
dermatitis to poison ivy)
TYPES OF ANTIGENS AND REACTIONS
● Antigens that trigger allergic reactions are called allergens. These low-molecular-weight substances can enter the body by being
inhaled, eaten, or administered as drugs.
● Hypersensitivity reactions can occur in response to different types of antigen, including environmental substances, infectious
agents, food, and self-antigens.
1. Environmental Substances
- Environmental substances in the form of small molecules can trigger several types of hypersensitivity reactions
- Dust can enter the respiratory tract, mimicking parasites, and stimulate an antibody response
- An immediate hypersensitivity reaction associated with IgE, such as rhinitis or asthma, can result
- If dust stimulates immunoglobulin G (IgG) antibody production, it can trigger a different type of hypersensitivity reaction,
such as farmer’s lung
- If small molecules diffuse into the skin and act as haptens, a delayed hypersensitivity reaction, such as contact dermatitis,
will result.
- Drugs administered orally, by injection, or on the skin can provoke a hypersensitivity reaction mediated by IgE, IgG, or T
lymphocytes.
- Metals (particularly nickel) and chemicals can also cause type I hypersensitivity reactions. Low-molecular-weight chemicals
usually act as a hapten by binding to body proteins or major histocompatibility complex (MHC) molecules. The complex of
antigen and MHC molecules is then recognized by specific T cells, which initiate the reaction.
2. Infectious agents
- Not all infectious agents are capable of causing hypersensitivity reactions
- The influenza virus can cause hypersensitivity that results in damage to epithelial cells in the respiratory tract
- Sometimes, an exaggerated immune response occurs. Influenza virus, for example, can trigger high levels of cytokine
secretion or what is called a cytokine storm
- In comparison, streptococci can cause a hypersensitivity reaction termed immune complex disease
3. Self-antigens
- Examples of self-antigens are cellular proteins, peptides, and enzyme complexes
- Very small immune responses to self-antigens is normal and occur in most people
- When these become an exaggerated response, however, or when tolerance to other antigens breaks down,
hypersensitivity reactions can occur.
4. Food allergies
- According to the National Institute of Allergy and Infectious Diseases (NIAID), food allergy (FA) is an important public health
problem that affects adults and children and may be increasing in prevalence
- Food allergy can cause severe allergic reactions and even death from food-induced anaphylaxis
- Despite the risk, there is no current treatment for FA; the disease can only be managed by allergen avoidance or treatment
of symptoms
- The diagnosis of FA may be problematic because nonallergic food reactions, such as food intolerance, are frequently
confused with FAs.
- Food intolerance differ from food allergies:
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o Food intolerance does not involve the immune system and does not cause severe allergic reactions
o Food allergy causes an immune system reaction that affects numerous organs in the body
o Allergic food reaction can also be severe or life-threatening
o Food intolerance symptoms are generally less serious and often limited to digestive problems
- The NIAID guidelines separate diseases defined as FA that include both IgE-mediated reactions to food (food allergies),
non–IgE-mediated reactions to certain foods (e.g., celiac disease), and mixed IgE and non-IgE disorders
TYPES OF HYPERSENSITIVITY REACTIONS
Four types of hypersensitivity reactions:

● Antibody-mediated and Immediate Hypersensitivity
- Type 1 Hypersensitivity
● Cell-mediated and Delayed Hypersensitivity
COMPARISON OF HYPERSENSITIVITY REACTIONS
TYPE I TYPE II TYPE III TYPE IV

Other name Anaphylactic Antibody-mediated Complex-mediated Cell-mediated
hypersensitivity cytotoxic hypersensitivity hypersensitivity
hypersensitivity
Immune IgE IgM / IgG IgM/ IgG T- Cells
mediator
Effector cells Basophil, Mast cell RBC, WBC, PLATELETS Host tissue cells Macrophages, T cells
Immune Release of Cytolysis due to Deposits of antigen Antigen-sensitized
mechanism mediators from antibody and antibody complexes Th1 cells release
mast cells and complement, that activates cytokines that recruit
basophils opsonization, or ADCC complement. macrophages
Neutrophil are and induce
recruited and release inflammation or
lysosomal enzymes activate cytotoxic T
that cause tissue cells to cause direct
damage cell damage
Antigen Heterologous Cell surface; Soluble: autologous or Autologous or
autologous or heterologous heterologous
heterologous
Complement NO YES YES NO
involvement
Cytokines Yes No Yes Yes
involvement
Examples 1.Anaphylaxis 1.Transfusion rxn 1. Serum sickness 1. Contact dermatitis
2. Hay fever 2. AIHA 2.Arthus reaction (poison ivy, nickel,
3.Food allergies 3. HDN 3. SLE mercury, copper
4.Asthma 4.GOODPASTURE’S 4.Rheumatoid rubber,
5.Drugs syndrome arthritis formaldehyde, hair
(e.g Penicillin) 5.Myasthenia gravis 5.Post streptococcal dyes, sunscreen
6.Rhinitis (most 6.Graves disease glomerulonephritis agents, disinfectants,
common) 7.ITP associated in SLE perfumes, and
7.latex allergy 6. Other autoimmune pesticides)
disorders 2. Tuberculin/ PPD
7.Neoplastic diseases test
3. Pneumonitis
4.Mantoux
5.anergy skin test
6.hypersensitivity
pneumonitis
7.Type 1 DM
8. GVHD
TYPE 1 HYPERSENSITIVITY
INTRODUCTION TO TYPE I HYPERSENSITIVITY:

 This is the only type of HPS that is mediated by IgE antibodies and mast cells.
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 The IgE response is the normal protective response against many metazoan parasites, which are too large to be
phagocytized or killed by other cytoplasmic mechanisms.
 Type I responses occur within minutes to hours of antigen re exposure.
 Commonly called allergic or immediate hypersensitivity reactions.
 Can be problematic when harmless environmental antigens (such as, pet dander or pollen) cause an exaggerated
immune response from the body. These responses are called atopic or allergic responses.
o Antigens that cause an allergic response can be called allergens.
MECHANISMS TYPE 1 HYPERSENSITIVITY
General Mechanism of Type I Hypersensitivity

 IgE receptors readily bind to Fc receptors (Fc epsilon RI or CD23) on the surface of mast cells and basophils
 Fc epsilon RI bind antigen-free Immunoglobulin (IgE), and the IgE-CD23 complexes function as antigen-specific
cell-surface receptors.
 Cross-linking of surface-bound IgE molecules generates intracellular signals via CD23.
 Leading to mast cell or basophil degranulation.
 And the release of vasoactive amines (e.g. histamine) and other inflammatory mediators.
 Histamine and other inflammatory mediators cause vascular endothelial cell junctions to loosen (vasodilation) and
increase in vascular permeability, resulting in fluid accumulation in the tissues (edema).
 Histamine also induces smooth muscle contraction in arterial and arteriole walls (vasoconstriction) to accelerate fluid
distribution from the central trunk of the body into peripheral tissues.
Immunologic Components Involved in Type I Hypersensitivity

 Allergens
 Mast Cells, Basophils
 IgE Antibody
 IgE-binding Fc Receptors
 Eosinophils (late phase)
A. Localized Reactions
- Type I reactions are often most pronounced in respiratory passages, intestinal walls and the skin, due
to the accumulation of mast cells in these tissues.
- Sites affected are where initiating antigen is most often encountered.
- Antigens that enter the body by inhalation localize primarily to the nasopharyngeal and bronchial
tissues, where smooth muscle contraction and vasodilation increase mucous production and the
constriction of respiratory passages. When these responses are combined, they can produce asthma.
- Allergens that contact other tissues may produce IgE-mediated inflammatory responses, causing
rashes, redness, and edema – the classic “wheal and flare” appearance.
- Food or ingested allergens primarily affect the GI tract.
- Site of entry for allergens = site of response
B. Systemic Reactions
- Injected allergens (e.g. venom or toxins), antigen may be disseminated by the bloodstream, resulting
in systemic inflammation.
- Site of entry for allergen is different from site of response
History of Anaphylaxis
 In 1902, at the request of and sponsorship of Albert I of Monaco, Charles Richet and Paul Portier investigated
jellyfish nematocyst toxin that sometimes induced a life-threatening response.
 They found out that initial injection of dogs with a small amount of toxin had little effect. However, when a second
injection of the same amount of toxin was administered several weeks later, the dogs suffered immediate shock and
even death.
 Termed anaphylaxis meaning “against protection” or “without protection”
 Anaphylaxis is characterized by vasoconstriction along with vasodilation resulting in severe fluid loss and leading to
shock.
Notice: The discovery of anaphylaxis by Richet and Portier went against the central dogma of
immunity/immunization.
History of Type I Hypersensitivity

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 In 1921, Carl Wilhelm Prausnitz and Heinz Kustner were the first researchers to show that a serum factor
was responsible for type I reactions.
 Serum from Kustner, who was allergic to fish, was injected to Prausnitz.
 A later exposure to fish antigen at the same site resulted in an allergic skin reaction.
- Redness and swelling
 This is called passive transmission of hypersensitivity
 It occurs when serum is transferred from an allergic individual to a non-allergic individual, and the second
individual is challenged at a later time with the specific allergen.
Two Major Phases:

 Sensitization phase
 Activation phase
NOTE: A late phase reaction may also occur in some individuals.
SENSITIZATION PHASE
 Also known as first exposure
 Environmental antigens are internalized and processed and are transported to the local lymphoid tissues
 APCs, such as dendritic cells, present antigen to Th cells
 Th cells bind to antigen and co-stimulatory molecules, and differentiate into Th2 cells
 Th2 cells release cytokines, such as Il-4 and IL-13 that stimulate B cells to produce IgE through antibody class
switching
 B cells produce IgE immunoglobulin
 Mast cells contain high-affinity receptors called Fc epsilon Ri, wherein the IgE antibody attached to
 The Fc epsilon RI bind the fragment crystallizable (Fc) region of the epsilon-heavy chain
 Once the IgE is bounded to the cell membrane, IgE serves as an antigen receptor on mast cells
 Fc epsilon RI = high-affinity
 Fc epsilon RII = low-affinity
NOTE: Mast cells are the principal effector cells of immediate HPS or type I HPS, due to their Fc epsilon RI
receptors.
ACTIVATION PHASE
 Also known as effect stage
 Upon second exposure to allergen
 Adjacent cell-bound IgE molecules cross-link by a bivalent or multivalent antigen, causing aggregation of
the surface Fc epsilon RI receptors
 Initiates complex intracellular signaling events involving multiple phosphorylation reactions, and influx of
calcium, and secretion of cytokines
 Increase in intracellular calcium triggers rapid degranulation of mast cells and basophils
- Degranulation is initiated by:
o Allergen immunologic cross-linkage of bound IgE
o Anaphylatoxins (C3a, C4a, C5a)
o Drugs
- NOTE: anaphylatoxins and drugs are non-immunologic factors
 Release of primary mediators, such as:
- Activated mast cell or basophil:
o Biogenic amines: histamine
o Lipid mediators: PAF1, PGD1, LTC4
o Cytokines: TNF
o Enzymes: trypase
- Eosinophil:
o Cationic granule proteins: major basic protein, eosinophil cationic protein
o Enzymes: eosinophil peroxidase
- Others: heparin, eosinophil chemotactic factor of anaphylaxis (ECF-A), neutrophil chemotactic factor
(NCF), proteases.
 The chemical mediators bind to receptors on target organs producing symptoms characteristic of an
allergic response
- Skin: wheal-and-flare reaction
- Respiratory Tract: contraction of SM in the bronchioles resulting in airway obstruction
LATE PHASE REACTION

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 Synthesis of secondary mediators, such as:
- PAF: platelet activating factor
- PGD2: prostaglandin D2
- LT: leukotrienes B4, C4, D4, E4
- Cytokines
 More potent than the primary mediators
 Can be seen in late-phase allergic reaction that is 6-8 hours after exposure to the antigen
 Eosinophils, neutrophils, Th2 cells, mast cells, basophils, and macrophages, exit the circulation and
infiltrate the allergen-filled tissue
Key effector cell: eosinophils
NOTE: The differing manifestations of type I HPS in different species or different tissues partly reflect
variations in primary and secondary mediators present
COMMON REACTIONS OF CHEMICAL MEDIATORS
Primary Mediators:
 Vasodilation – increase vascular permeability
 Excessive mucus production
 Smooth muscle contraction
Secondary Mediators:
 Tissue damage
 Eosinophilia – cytokines such as IL-5  Th2 cell stimulation  increase production of eosinophils
 Tissue remodeling – thickening of SM, changes in CT, blood/lymphatic vessels, mucus glands, nerves
ENVIRONMENTAL/GENETIC INFLUENCES ON TYPE I HYPERSENSITIVITY
ENVIRONMENTAL
Atopy:
 Refers to an inherited tendency to respond to naturally occurring inhaled and ingested allergens with
continued production of IgE
 A hereditary predisposition to the development of immediate hypersensitivity reactions against common
environmental antigens
 Allergies with strong familial or genetic tendency
 T cells from the blood of atopic patients respond to allergens in vitro by inducing cytokines produced by
Th2 cells (IL-4, IL-5, IL-13), rather than cytokines produced by Th1 (IFN-gamma, IL-2)
- Immunologic hallmark: infiltration of affected tissue by Th2 cells
 Farm Effect:
- Utero or early life exposure to the diverse microbial populations in a farming environment provides protection
against allergies by inducing development of Treg cells and by directing the immune system toward beneficial
Th1 responses and away from Th2 atopic reactions
 Increased in allergy prevalence may be due to increase hygiene practices and use of antibiotics, with consequent
decrease in exposure to microbes
 Exposure to stress, variations in physical factors, and contact with environmental pollutants can intensify clinical
manifestations of allergy in susceptible individuals
GENETICS
 Chromosome 5q – linked to a region that encodes a variety of cytokines (Il-3, IL-4, IL-5, IL-9, IL-13, and GM-
CSF)
 Chromosome 11q – linked to a region that encodes the B-chain of the high-affinity IgE receptor.
 Several hundred genes are associated with susceptibility to developing allergies
o Alteration of protective barrier of the body
o Antigen recognition by innate immunity – CD14 and TLRs
o Cytokine production
o Differentiation of Tcells  Th1, Th2, Treg cells
o HLA class II genes  allergy and asthma
o HLA-D  antigen presentation and may influence the tendency to respond to specific allergens
6
o Modulation of inflammatory response can influence long-term consequences of allergies by affecting the process
of tissue remodeling and repair
Note: Fc epsilon RI – high affinity; Fc epsilon R2 – low affinity
CLINICAL MANIFESTATIONS AND EXAMPLES OF TYPE 1 HYPERSENSITIVITY
 Clinical manifestations are common, especially in developed countries

 40% of the world’s population has allergic sensitization to common environmental antigens
 5th leading cause of chronic disease in all ages
 3rd leading cause of chronic disease in children
ANAPHYLAXIS
 Most severe type of allergic response
 Acute reaction BUT it simultaneously involves multiple organs
 Severe systemic response caused by the release of inflammatory mediators from mast cells and basophils
- Bronchoconstriction
- Vasomotor collapse
 Most common cause:
- Drugs (systemic PEN): acting as haptens that may become immunogenic by combining with host cells or
proteins
o Examples: penicillin, cephalosporin, and sulphonamide antibiotics, and muscle relaxants
- Insect stings: hymenoptera (common hornet, yellow jacket, yellow hornet, paper wasp)
o Contain enzymes such as phospholipases and hyaluronidases and other proteins that can elicit an IgE
antibody response
- Latex
o Latex is a milky sap produce by the rubber tree Hevea brasinliensis. Latex-related allergic reactions can
complicate medical procedures, for example, internal examinations, surgery, and catherization. Medical and
dental staff may develop occupational allergy through use of latex gloves
 Most common organ systems involved:
- GI tract: abdominal pain, hyperperistalsis, nausea, vomiting, diarrhea
- Oral surface: edema of lips and tongue
- Respiratory tract: upper airway obstruction, angioedema of tongue, bronchospasm, rhinitis, cough, wheezing,
sneezing, congestion
- Cutaneous: erythema, flushing, urticaria, pruritus
- Cardiovascular: faintness, hypotension, arrythmias, hypovolemis shock, syncope, chest pain
- Ocular: periorbital edema, erythema, conjunctival erythema, tearing
- Genito-urinary: uterine cramps, urinary urgency or incontinence
 Multiple exposure to antigen result in additional accumulation of IgE on the surface of the amst cells and basophils
 Death may result from asphyxiation because of upper-airway edema and congestion, irreversible shock, or a
combination
ALLERGIC RHINITIS
 Watery nasal discharge, sneezing
 The most common form of atopy
 Affects 10-30% of the world’s population
 Hay fever – seasonal allergic rhinitis, that is triggered by tree and grass pollens
 Seasonal: symptoms of seasonal allergic rhinitis can occur in spring, summer and early fall. They are usually caused
by allergic sensitivity to airborne mold spore or to pollens from trees, grass, and weeds
 Perennial: people with perennial allergic rhinitis experience symptoms year-round. It is generally caused by dust
mites, pet hair or dander, cockroaches, or mold.
ALLERGIC ASTHMA
 Derived from the Greek work “panting” or “breathlessness”
 Reversible airway obstruction often caused by the release of inflammatory mediators from mast cells upon encounter
with allergen
 Loosening of tight junctions in the bronchiole epithelium, increased capillary permeability, and spasmatic contraction
of smooth muscle surrounding the bronchi
 Temporary decrease in size of the bronchial lumen, results in shortness of breath
 Bronchospasms
 The most common asthma
o 90% of kids with childhood asthma have allergies, compared with about 50% of adults with asthma
o Symptoms show up after you breathe in allergens.
o It usually gets worse after you exercise in cold air or after breathing smoke, dust, or fumes
o Since allergens are easy to inhale, avoid triggers as much as possible
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FOOD ALELRGIES
 When the body reacts unusually to specific foods
 Most common foods that induce an allergic response:
- Cow’s milk, eggs, nuts, soy, wheat, fish and shellfish
 The most common forms of immune-mediated adverse reactions to foods always are characterized by the
development of IgE against food allergens. It can be accompanied by inflammation, induced by cellular components,
and mediated by T cells and eosinophils.
 Patients with IgE-associated food allergy can be identified based on the detection of food allergen-specific IgE in
serum and body fluids, and by measuring IgE-mediated cellular and in vivo responses
 It is not only common, but often is a serious and life-threatening health conditions
 Requires accurate diagnosis
 Can be confused with food intolerance
- Food intolerance: eating it can make you feel uncomfortable but no immune response or allergic response
- Food allergy: eating certain foods induces immune response
 Class 1 food allergen: are oral allergens that cause sensitization via the GI tract
 Class 2 food allergen: food allergens are aeroallergens that cause sensitization via the respiratory tract. Immune
response against these allergens can cross-react with homologous food allergens
Most common food-induced allergic reactions (World Allergy Organization, 2017):
PATHOLOGY DISORDER KEY FEATURES MOST COMMON CAUSAL

FOODS
IgE-mediated
(acute onset)
Acute Food commonly causes acute (20%) Primarily ”major allergens”
urticaria/angioedema but rarely chronic urticaria
Contact urticaria Direct skin contact results in lesions. Multiple
Rarely this is due to direct histamine
release (non-immunogenic
Anaphylaxis Rapidly progressive, multiple organ
Any but more commonly
system reaction can include peanut, tree nuts,
cardiovascular collapse shellfish, fish, milk, and
egg
Food-associated, Food triggers anaphylaxis only if Wheat, shellfish, and
exercise-induced ingestion followed temporally by celery most described
anaphylaxis exercise
Oral allergy syndrome Pruritus, mild edema confined to Raw fruit/vegetables.
(pollen-associated oral cavity. Uncommonly progresses Cooked forms tolerated.
food allergy beyond mouth (~7%) or anaphylaxis Examples of relationships:
syndrome) (1-2%). May increase following birch (apple, peach, pear,
pollen season carrot, ragweed (melons))
Immediate Immediate vomiting, pain Major allergens
gastrointestinal
hypersensitivity
ALLERGIC URTICARIA
 Also known as hives
 Appear within minutes after exposure to the allergen
 Wheals (dermal edema), erythema (redness), angioedema (severe local swelling)
 Very common when encountered in food allergy
ATOPIC DERMATITIS
 Also known as eczema
 Can take on a variety of forms: erythematous, oozing vesicles to thickened, scaly skin, depending on the
stage of activity and age of individual
 It is a chronic, itchy skin rash that usually develops during infancy, and persists during childhood
 Chromosome: 1q21 – epithelium-related genes  mutations in filaggrin (key protein in epidermal
differentiation)
TESTING OF TYPE 1 HYPERSENSITIVITY
Laboratory test:
 RIST – for quantitating total IgE
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 RAST (radioallergosorbent test) – for quantitating allergen- specific IgE
 The RIST AND RAST test are in-vitro tests
SKIN PRICK TEST (SPT)

 Placing a drop of a solution containing possible allergen on the skin
 A series of scratches or needle pricks allow the solution to enter the skin
 (+): red, raised, itchy area
CONS:
 Not diagnostic of food allergy
 This procedure carries the risk of triggering a systemic reaction (e.g., anaphylactic reaction) or initiating a
new sensitivity
 About 50-60 percent of all SPTs yield “false positive” results, meaning that the test shows positive even
though you are not really allergic to the food being tested.
 It involves putting a drop of liquid onto your forearm that contains a substance you may be allergic to.
The skin under the drop is then gently pricked. If you're allergic to the substance, an itchy, red bump will
appear within 15 minutes.
PATCH TEST
 Used for the evaluation of contact food allergies
 Involves taping a patch that has been soaked in the allergen solution to the skin for 24-72 hours
 Used to detect contact dermatitis
 Patch tests don't use needles. Instead, allergens are applied to patches, which are then placed on
your skin. During a patch test, your skin may be exposed to 20 to 30 extracts of substances that can cause
contact dermatitis
IMMUNO CAP RAST

 Considered to be the gold standard for the analysis of allergen- specific IgE
 RAST and ELISA can test for pet allergies and food allergies. Blood tests like RAST and ELISA can test for a
range of allergies, including food allergies, drug allergies, seasonal allergies, and pet allergies.
Which is more accurate skin test or blood test?

 Generally speaking, skin tests are more sensitive than blood tests, meaning they are more likely to
detect allergies that a blood test may miss
 Skin tests also require less wait time, as results are typically delivered in 15-20 minutes, rather than the
one to two week wait time of blood tests.
Does allergy cured permanently?

 No, but you can treat and control your symptoms. You'll need to do all you can to prevent being exposed
to things you're allergic to – or example, staying inside on days when the pollen count is high, or enclosing
your mattress with a dust-mite-proof cover. Allergy medicine can also help.
TREATMENT OF TYPE 1 HYPERSENSITIVITY
 Avoidance of allergens is the first line of defense.

 Pharmacological therapy is necessary to relieve acute symptoms, control chronic allergy manifestations,
and, in some cases, modulate the immune response to the allergen.
TREATMENT/s:
 DRUGS are used to treat immediate hypersensitivity vary with the severity of the reaction.
Drug therapy
 Epinephrine(adrenaline)- stimulates both alpha adrenergic and beta-adrenergic receptors decrease
vascular permeability
 This medication is used in emergencies to treat very serious allergic reactions to insect stings/bites, foods,
drugs, or other substances
 Epinephrine acts quickly to improve breathing, stimulate the heart, raise a dropping blood pressure,
reverse hives, and reduce swelling of the face, lips, and throat
 Epinephrine is in a class of medications called alpha- and beta-adrenergic agonists (sympathomimetic
agents).
9
Antihistamine
 Best used as a pre-exposure drug
 Block specific histamine receptors
 Not very useful in asthma because histamine is not an important allergic mediator released by mast cell in
the lungs.
 Antihistamines reduce or block histamines, so they stop allergy symptoms. These medicines work well to
relieve symptoms of different types of allergies, including seasonal (hay fever), indoor, and food allergies.
 Antihistamines have been used for years to treat allergy symptoms. They can be taken as pills, liquid, nasal
spray, or eye drops.
 Over-the-counter (OTC) antihistamine eye drops can relieve red itchy eyes, while nasal sprays can be used
to treat the symptoms of seasonal or year-round allergies.
Leukotriene antagonist (montelukast)

 Reduces amount of airway inflammation in asthma
 Leukotrienes (LTs) are lipid mediators that play pivotal roles in acute and chronic inflammation and allergic
diseases.
 They exert their biological effects by binding to specific G-protein-coupled receptors
 Each LT receptor subtype exhibits unique functions and expression patterns.
Corticosteroid
 Often given topically
 Corticosteroids are a form of steroids used to treat swelling and inflammation from allergies, as well
as allergic asthma
Desensitization (Immunotherapy)
 Applicable if only one allergen is incriminated
 If a patient has a history of life-threatening conditions, and if other treatment alternatives are
unsatisfactory, desensitization is used to prevent anaphylaxis resulting from insect stings (e.g., yellow
jackets)
 It aims to do exactly that: make the immune system less sensitive to the allergen by allowing it to "get
used to" it. An allergy is an exaggerated reaction to a substance that is actually harmless.
 Desensitization is the most important to treat allergy
 Because this desensitization is the very important process whereby cells decrease their sensitivity to a
particular neurotransmitter to prevent saturation of the system.
Mechanishm of Desensitization:
 Downregulation of the Th2 Cells
 Upregulation of Th1 cytokines
 Induction of Treg cells
Administration of increasing concentration of allergen:
1. DOWNREGULATION – Th2 (DECREASES)

- Type cytokines (interleukin- 4 , interleukin- 5)
- IgE
- Eosinophils
- Mast cells
- Basophils
- Late-phase reaction
- Immediate hypersensitivity
NOTE: Downregulation and desensitization can be metaphorically understood as phenomena that increase the

"static stability" of receptor systems. They render the systems more "resilient" and thereby enable the
response to bounce back in a rapid fashion following an external perturbation.
2. UPREGULATION - Th1 (INCREASES)

- Type cytokines (interferon- y, interleukin-12 ) IgG
- Upregulation: An increase in the number of receptors on the surface of target cells, making the cells
more sensitive to a hormone or another agent.
- IL-12 is predominantly produced by dendritic cells, monocytes, and macrophages, and to a lesser
extent by B-cells.
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Upregulation
 Regulatory T cells (INDUCES)- interleukin -10 transforming growth factor beta
 Allergen- specific hyporesponsiveness.
 Allergens that trigger the production of antibodies called Immunoglobulin E.
 IgE: these antibodies travel to cells that release chemicals, causing an allergic reaction
 This reaction usually causes symptoms in the nose, lungs, throat, or on the skin.
THERAPUETIC OPTIONS FOR ALLERGY

 Removal or avoidance of allergic triggers
 H1 blockers
 Mast cell stabilizers
 Anti-inflammatory corticosteroid and leukotriene inhibitors
 Immunotherapy
TREATMENT OF ASTHMA:
 Omalizumab: is a specific for IgE
NOTE:
 There are many good asthma treatments, but most require a prescription
 These medications include inhaled steroids, which fight inflammation, and bronchodilators, which open up
your airways.
 If traditional treatments don't help your allergic asthma, Xolair, an injectable medication that reduces IgE
levels, may help.
 XOLAIR is the only medication specifically designed to treat moderate to severe persistent allergic asthma
in patients 6 years of age and older who are uncontrolled with inhaled corticosteroids. ALSO KNOWN AS
OMALIZUMAB
 Histamine does not play a significant role in bronchial constriction, therefore antihistamines ( H1 receptor
antagonist) are not used to treat asthma.
ALLERGY IMMUNOTHERAPY
 One potentially very beneficial approach to the treatment of allergy is to used immunotherapy
 Used for allergens that are hard to avoid
 Subcutaneous injection of small amounts of allergen in gradually increasing doses
 Increases weekly or biweekly by 2 or less times until the maximum tolerated dose is reached
 Patients observed for 30 minutes during dose escalation due to risk of anaphylaxis
 Alternative routes of administration include sublingual and oral
 Shifts response away from Th2/ IgE and increasing the activity of regulatory T – cells
NOTE: Allergy shots are regular injections over a period of time — generally around three to five years — to
stop or reduce allergy attacks. Allergy shots are a form of treatment called immunotherapy. Each allergy shot
contains a tiny amount of the specific substance or substances that trigger your allergic reaction
 Also known as antibody mediated-cytotoxicity hypersensitivity

 It refers to an antibody-mediated immune reaction in which antibodies IgG and IgM are directed against cellular or
extracellular matrix antigens with resultant cellular destruction, functional loss, or damage to tissues
MECHANISMS TYPE 2 HYPERSENSITIVITY
Type 2 hypersensitivity reaction

 A type II hypersensitivity is said to occur when damage to the host tissues is caused by cellular lysis induced by the
direct binding of antibody to cell surface antigens. While the antibodies involved in type I HS are of the IgE isotype,
those involved in type II HS reactions are mainly of the IgM or IgG isotype.
 It includes IgG and IgM antibodies (directed against cell antigens found on cell surfaces)
 These antigens may be altered as:
1. Self-antigens
2. Heteroantigens
 Binding of antibody to cell have three major effects:
1. Cell can be destroyed
2. Function of cell can be inhibited
3. Function of cell can be increased above normal
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 Cell Damage occur in several different mechanisms, some of which involve complement as well as antibodies
1. Activation of classical pathway of complement can lead to the formation of membrane attack complex and cell
lysis
2. Coating of the cell surface by antibodies can promote opsonization and subsequent phagocytosis of the cell
3. Cell damage can result from mechanism of antibody dependent cellular cytotoxicity (ADCC)
 Type II hypersensitivity reaction involves antibody mediated destruction of cells. It is also known as cytotoxic
reaction.
 The killing of cell can occur by one of the three mechanisms:
1. Complement mediated cell lysis
2. Antibody dependent cell mediated cytotoxicity (ADCC)
3. Opsonization
Complement mediated lysis of cell:
 Complement system is a system of lytic enzyme which are usually inactive in blood.
 Enzymes of complement system are activated by antigen-antibody complex.
 When antibody binds to antigen (microorganism or RBC) they form Ag-ab complex.
 Ag-ab complex can activate complement system by three different mechanism-classical pathway, alternate pathway
and lectin pathway.
 Activated complement proceeds in cascade mechanism.
 When complement is activated on the surface of cell (RBC) it causes lysis of cell.
Antibody dependent cell mediated cytotoxicity (ADCC):

 Antibody binds with antigen by its Fab portion. However, Fc region of antibody has receptor on cytotoxic cells. So,
antibody cross link target cell (microorganism or RBC) with cytotoxic cells and promote killing.
 Most cytotoxic cells contain storage of hydrolytic and digestive enzymes. These enzymes are released on the surface
of target cell (MOs or RB or target cell), killing them.
 Here antibody itself does not kill or destroy cell but rather mediate killing by presenting antigen to cytotoxic cell.
Similarly, cytotoxic cell depends upon antibody to bind antigen. So this mechanism is known as Antibody dependent
cell mediated cytotoxicity.
 Mediated though binding of IgG antibody to its corresponding antigen on target cell and Fc receptors on macrophages
or natural killer cells
 This binding stimulates the release of cytotoxic enzymes that destroy the cell
Opsonization:
 When antigen enters into host body, antibodies are produced
 Antibody binds to antigen through Fab region. Fc region of antibody remains free.
 Phagocytic cells such as Neutrophils, macrophages and monocytes have receptors that can bind to Fc region of
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antibody. The receptor is known as FcR.
 In this case antibody molecule directly cross links antigen (Microorganism or RBC or target cell) with phagocytic
cells. This cross-linkage activates phagocytic cells and increases the rate of phagocytosis.
 This increased rate of phagocytosis by binding of antibody to antigen is called Opsonization.
CLINICAL MANIFESTATIONS AND EXAMPLES OF TYPE 2 HYPERSENSITIVITY
Clinical Examples involved in destruction of cells in Type II Hypersensitivity include:

A. Blood transfusion reactions
B. Hemolytic disease of newborn
C. Autoimmune hemolytic anemia
A. Blood transfusion reactions

- Acute transfusion reactions present as adverse signs or symptoms during or within 24 hours of a
blood transfusion. The most frequent reactions are fever, chills, pruritus, or urticaria, which typically resolve
promptly without specific treatment or complications.
- Examples of cell destruction resulting from antibodies combining with heteroantigens
- Major groups involved in transfusion reactions include the ABO, Rh, Kell, Duffy, and Kidd systems
- ABO Blood Group is important in considering transfusions
- Anti-A and Anti-B antibodies are naturally occurring antibodies (isohemagglutins)
- ABO blood transfusion reaction is an example of type II hypersensitivity reaction. Human RBCs contains A and/or B antigen
as major antigen on the surface of RBC. Other minor antigens such as Rh, Kell, Duffy etc. are also present. Antibodies to ABO
antigen are called isohemagglutinin and are usually of IgM class whereas antibodies to other minor antigen are of IgG class.
- An individual with blood group A recognizes B antigen like epitope (blood group B) as foreign and produces
isohemagglutinin (antibodies). The same individual does not produce antibodies to A antigen as it is similar to self-antigen,
so that state of tolerance exists.
The extent of reaction depends on the following factors:

 Temperature at which the antibody is most active
 Plasma concentration of the antibody
 Immunoglobulin class involved
 Extent of complement activation
 Density of antigen on RBC
 Number of RBCs transfused
Acute Hemolytic Transfusion Reactions

 Acute hemolytic transfusion reactions are usually caused by ABO incompatibility. This potentially fatal complication
occurs in about 1 in 30,000 transfusions
 As little as 20 to 30 mL of incompatible RBCs can cause agitation, nausea and vomiting, dyspnea, fever, flushing,
hypotension, tachycardia, and hemoglobinuria.
 Occur within minutes or hours after receipt of incompatible blood
Intravascular hemolysis
 Intravascular hemolysis is the destruction of red blood cells in the circulation with the release of cell contents into the
plasma
 Mechanical trauma from a damaged endothelium, complement fixation and activation on the cell surface, and
infectious agents may cause direct membrane degradation and cell destruction.
 Occurs because of complement activation, resulting in release of hemoglobin and vasoactive and procoagulant
substances into the plasma
 This may induce disseminated intravascular coagulation (DIC), vascular collapse, and renal failure
 Symptoms in patient may include chills, fever, nausea, lower back pain, tachycardia, shock and hemoglobin in urine
B. Hemolytic disease of newborn

- (HDN) is a blood disorder in a fetus or newborn infant. In some infants, it can be fatal. Normally, red blood
cells (RBCs) last for about 120 days in the body. In this disorder, RBCs in the blood are destroyed quickly and
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thus do not last as long.
- it appears in infants whose mothers have been exposed to blood-group antigens on baby’s cells that differ from
their own
- severe HDN is called Erythroblastosis fetalis (most common antigen involved here is the D antigen, member of
Rh group)
- HDN is caused by ABO incompatibility (more common)
- The disease is milder, possibly because antibodies are neutralized by A and B antigens found in fetal tissues or it
is because A and b antigens on fetus RBCs are more poorly developed or reduced in number
- Other antibodies associated with HDN include anti-c, anti-C, and anti-E
- Exposure usually occurs during birth process when fetal cells leak into the mother’s circulation (first child is
unaffected, second and later children are affected due to anamnestic response)
- Hemolytic disease of newborn develops when maternal IgG antibodies specific for fetal blood group crosses placenta and
destroy fetal RBCs.
- The consequences of such transfer of antibody can be minor, serious or lethal to fetus.
- Serious hemolytic diseases of new born develops when Rh –ve mother conceive Rh +ve fetus, which causes
erythroblastosis fetalis.
- - During pregnancy fetal RBCs are separated from mother’s circulation by a layer of cell in placenta called trophoblast.
During her 1st pregnancy with Rh+ve fetus, mother circulation is not exposed to enough fetal RBC to activate Rh Specific B
cells for antibody production.
- At the time of delivery, large amount of fetal umbilical cord blood enter to mother’s circulation. These fetal blood activates
mother Rh specific B cells resulting in production of plasma cell and memory cell. The plasma cell produce IgM antibodies
which binds and destroy fetal RBCs from mother’s circulation but the memory cell remains which threat any subsequent
pregnancy with Rh+ve fetus.
- Activation of memory cells in subsequent pregnancy with Rh+ve fetus causes production of IgG antibodies which can cross
placenta and destroy fetal RBCs.
- Mild to severe anemia develops in fetus and sometime fetal. The conversion of hemoglobin to billirubin produces additional
threat to new born because billirubin may accumulate in brain and damage it.
- This hemolytic disease of new born can be prevented by injecting preformed antibodies against Rh antigen to mother at
around 28 weeks of pregnancy and within 24-48 hours of 1 st The antibodies marketed as Rhogam. These antibodies bind to
RBCs of fetus in mother circulation and clear before B cell activation.
C. Autoimmune Hemolytic Anemia

- Autoimmune hemolytic anemia is a rare red blood cell disorder and an immune disorder
- It happens when the body produces antibodies that destroy the red blood cells. Hemolytic anemia develops when
there are not enough red blood cells because the body destroys them sooner than it should.
- It is an example of type II hypersensitivity reaction directed against self-antigens because individuals with these
disease form antibodies to their own RBCs
- It has been estimated to occur in 1 in 50,000 to 80,000 individuals
- Symptoms include:
1) Malaise
2) Lightheadedness
3) Weakness
4) Unexplained fever
5) Pallor
6) Possibly mild jaundice
Such antibodies can be categorized into two groups:
1. Warm Reactive Antibodies

- Warm antibody hemolytic anemia is the most common form of autoimmune hemolytic anemia. It is defined by
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the presence of autoantibodies that attach to and destroy red blood cells at temperatures equal to or greater than
normal body temperature.
- Reacts at 37 degree Celsius
2. Cold Reactive Antibodies

- Cold agglutinins are antibodies, typically IgM, that are acquainted with and then binding the antigens on red
blood cells, typically antigens “I” or “i” on the RBC surface
- In the environment in which the temperatures are lower than normal core body temperature and, thus, ends up
leading to agglutinations of the red blood cells and hemolysis reaction occurring outside the vessels (extra-
vessels), resulting in anemia without hemoglobinuria in ordinary cases.
- Reacts below 30 degree Celsius
Warm Autoimmune Hemolytic Anemia

 Warm autoimmune hemolytic anemia (WAHA) is an autoimmune disorder characterized by the premature destruction
of healthy red blood cells (hemolysis)
 Autoimmune diseases occur when one's own immune system attacks healthy tissue.
 It accounts for more than 70% of autoimmune anemias
 It is characterized by formation of IgG antibody
 Reacts mostly at 37 degree Celsius
 Some of these antibodies may be primary with no other disease association
 Others may be secondary to another disease process
 Associated disease may include:
A. Viral
B. Respiratory infections
o such as infectious mononucleosis, cytomegalovirus, chronic active hepatitis
C. Immunoproliferative diseases
o such as chronic lymphocytic leukemia, and lymphomas
Idiopathic Autoimmune Hemolytic Anemia

 The underlying cause of antibody production is unknown
 Idiopathic autoimmune hemolytic anemia is a form of autoimmune hemolytic anemia.
 Autoimmune hemolytic anemia (AIHA) is a group of rare but serious blood disorders. They occur when the body
destroys red blood cells more rapidly than it produces them. A condition is considered idiopathic when its cause is
unknown.
Second possible effect of Type 2 Hypersensitivity:

 Cell surface antibody can inhibit the function of a cell
 Occurs when antibody blocks the binding of psychological ligand to its receptor and result to dysfunctional of cell
 Examples: Myasthenia gravis, Graves’ disease
Myasthenia gravis Graves’ Disease
1.
Myasthenia gravis
- Myasthenia gravis (MG) is a neuromuscular disorder that causes weakness in the skeletal muscles, which are the
muscles your body uses for movement. It occurs when communication between nerve cells and muscles becomes
impaired.
- Symptoms: Muscle weakness
- Features are:
o drooping of eyelid
o weakness of arms and legs
o change of voice
o swallowing difficulty
2. Graves’ disease
- Graves' disease is an immune system disorder that results in the overproduction of thyroid hormones
(hyperthyroidism).
- Although a number of disorders may result in hyperthyroidism, Graves' disease is a common cause.
- Thyroid hormones affect many body systems, so signs and symptoms of Graves' disease can be wide ranging.
- An autoimmune disorder of thyroid gland
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Thyroid – Stimulating Hormone
 Thyroid stimulating hormone is produced and released
into the bloodstream by the pituitary gland.
 It controls production of the thyroid hormones, thyroxine
and triiodothyronine, by the thyroid gland by binding to
receptors located on cells in the thyroid gland.
 Hormone produced by pituitary gland in brain
 Binds to TSH receptors and stimulates thyroid cells to
produce hormones that increase metabolism
Hyperthyroidism
 Hyperthyroidism is a condition in which an overactive
thyroid gland is producing an excessive amount of thyroid hormones that circulate in the blood
 "Hyper" means "over" in Greek
 Thyroid hormones include thyroxine (T4) and triiodothyronine (T3)
- T3 is actually the most active thyroid hormone.
 Treatments: Radioactive iodine therapy; Thyroidectomy
 Symptom associated with increased metabolism
Goodpasture’s syndrome
 Autoimmune diseases are frequently Type II Hypersensitivity. Goodpasture's Syndrome is an example
 In Goodpasture's Syndrome, IgG and complement attack the kidney resulting in damage to the kidney basement
membrane.
 Goodpasture's syndrome is a rare disease that affects the lungs and kidneys.
 A combination of factors is associated with this disease, including the presence of an inherited component and
exposure to certain chemicals.
 Goodpasture's syndrome can be treated with immunosuppressive drugs and a process called plasmapheresis (the blood
plasma is cleaned) to remove the harmful antibodies from the blood.
 The syndrome may occur for variable time periods, from a few weeks to several years. Generally, this disease does
not lead to permanent lung damage, but kidney damage may be long -lasting.
 If the person develops kidney failure, then dialysis or kidney transplantation may be necessary
Pemphigus vulgaris
 Pemphigus is another Type II autoimmune disease
 Antibodies are produced against chromosomal proteins, skin, and mucous membranes which results in blistering.
 Sores and blisters almost always begin in the mouth. Auto -antibodies attack the “glue," which holds skin cells
together, called desmogleins, and the skin can tear easily in this disease.
Thrombocytopenia
 Antibodies can develop against red blood cells and produce anemia (low red blood cell count). Body temperature can
affect the reactivity of these antibodies
 Warm Antibody Hemolytic Anemia is an autoimmune disorder characterized by the premature destruction of red
blood cells by the body's natural defenses against invading organisms (antibodies)
 Normally, the red blood cells have a life span of approximately 120 days before they are removed by the spleen
 In an individual affected with Warm Antibody Hemolytic Anemia, the red blood cells are destroyed prematurely and
bone marrow production of new cells can no longer compensate for their loss
 The severity of the anemia is determined by the time the red blood cells are allowed to survive and by the capacity of
the bone marrow to continue new red blood cell production
 Immune Hemolytic Anemias are subdivided by the optimal temperature at which the antibodies destroy red blood
cells
 As their names imply, Warm Antibody Hemolytic Anemia occurs at temperatures of 37 degrees centigrade or higher
while Cold Antibody Hemolytic Anemia usually occurs at approximately 0 to 10 degrees
 In addition, platelets can also be attacked in Type II Hypersensitivity. This can lead to thrombocytopenia. Without
sufficient platelets, continued bleeding can occur.
TESTING OF TYPE 2 HYPERSENSITIVITY
 Coombs Test (Hemolytic Disease of the Newborn)

- The direct Coombs test detects maternal anti-D antibodies that have already bound to fetal RBCs. First, a sample
of fetal RBCs is washed to remove any unbound antibody (Ig). When the test antibodies (anti-Ig) are added, they
agglutinate any fetal RBCs to which maternal antibodies are already bound. This is called the direct Coombs test
because the anti-Ig binds "directly" to the maternal anti-D Ig that coats fetal RBCs in HDN.
 Coombs Test (Hemolytic Transfusion Reaction)

- When antibodies to the human globulin fractions are added to the patient’s blood, they will bind to the surface
antibodies and complement particles and cause the donor RBCs to agglutinate. The results are quantified on a
scale from 1+ to 4+ indicating a positive test.
 Coombs Test (Autoimmune Hemolytic Anemia)

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- Autoimmune hemolytic anemia is diagnosed by detection of autoantibodies with the direct Coombs test. The
direct Coombs test may be positive in the absence of autoimmune hemolytic anemia, and thus should be ordered
only in the proper clinical setting. A false-positive direct antiglobulin test may result from the presence of
clinically insignificant antibodies.
 Indirect Coombs’ Test

- Used in the cross matching of blood to prevent a transfusion reaction.
- Determine the presence of a particular antibody in a patient or type patient RBCs for specific blood
group antigens.
- Detects in vitro binding of antibody to RBCs.
 Direct fluorescence examination of a renal tissue biopsy for Good Pasture’s syndrome
- In Goodpasture syndrome, renal biopsy under a light microscope shows crescentic glomerulonephritis.
Immunofluorescence shows the linear deposition of IgG with a complement along the basement membrane. In
pemphigus vulgaris, histopathology shows suprabasal clefting and the "tombstone" appearance of the basal cells.
Immunofluorescence shows intercellular deposition of antibodies against IgG and C3.
 There is no cure for these diseases

 The treatment aims at symptom control only
 Because of the pathogenesis of these diseases are antibody in origin, a lot of treatment options are aimed at that.
 Depending on the severity of the hypersensitivity reaction, different treatment approaches are applied. Treatment
options, either given alone or in combination, include the following:
Steroids
 These drugs include prednisolone, dexamethasone, etc. In type II hypersensitivity diseases, sometimes high dose
steroids are used. Depending on the diseases, steroid could become a long-term medication. In such cases, long term
use will need medical supervision for monitoring of potential side effects.
There are other treatment methods all aiming at altering the body’s immune response, this includes:
 Intragam infusion: this is infusing the body with antibodies. There are many potentially severe side effects due to
this, hence it must be administered under specialist supervision.
 Plasmapheresis: this is removing the blood autoantibodies
 Other drugs such as interferon, cyclophosphamide, cyclosporin
 The treatment also includes anti-inflammatory and immunosuppressive agents
 It is also called ‘immune-complex’

 Occurs when there is accumulation of immune complexes that have not been adequately cleared by innate immune
cells
 Giving rise to an inflammatory response and attraction of leukocytes
 The most common diseases involving a type III hypersensitivity reaction are serum sickness, post-streptococcal
glomerulonephritis, systemic lupus erythematosus, farmers' lung (hypersensitivity pneumonitis), and rheumatoid
arthritis.

HISTORY
 Type III hypersensitivities are immune-complex reactions that were first characterized by Nicolas Maurice Arthus
(1862–1945) in 1903.
 To produce antibodies for experimental procedures, Arthus immunized rabbits by injecting them with serum from
horses. However, while immunizing rabbits repeatedly with horse serum, Arthus noticed a previously unreported and
unexpected localized subcutaneous hemorrhage with edema at the site of injection.
 This reaction developed within 3 to 10 hours after injection.
 This localized reaction to non-self serum proteins was called an Arthus reaction
 An Arthus reaction occurs when soluble antigens bind with IgG in a ratio that results in the accumulation of antigen-
antibody aggregates called immune complexes.
UNIQUE CHARACTERISTICS OF TYPE III HYPERSENSITIVITY

 Antibody excess (primarily IgG)
 Coupled with a relatively low concentration of antigen.
 Resulting in the formation of small immune complexes that deposit on the surface of the epithelial cells lining the
inner lumen of small blood vessels or on the surfaces of tissues.
MECHANISMOF TYPE 3 HYPERSENSITIVITY
● When antibody combines with its specific antigen, immune complexes are formed.
● Normally, they are promptly removed, but occasionally, they persist mostly due to their small size and are deposited in tissues
resulting into several disorders.
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● Such complexes are more commonly found to be deposited in joints, kidneys and blood vessels causing arthritis, nephritis and
vasculitis respectively while less commonly on other organs leading to organ dysfunction.
● Wherever immune complexes are deposited, they activate the complement system, and macrophage and neutrophils are
attracted to the site, where they cause inflammation leading to tissue injury.
● Type III hypersensitivity is primarily mediated by antibodies of the IgG and IgM classes which combine with soluble antigen that
are not bound to cell surfaces. The antigens may be self or foreign (i.e., microbial). Tissue damage is caused mainly by
complement activation and release of lytic enzymes from neutrophils.
● The reaction can take hours, days, or even weeks to develop, depending on whether or not there is immunological memory of
the precipitating antigen. The response can also become chronic, particularly in autoimmune reactions, where antigen persists.
● Type III hypersensitivity as in other cases of hypersensitivity occur when the mechanism of self-tolerance is breached and some
self-reactive immune cells are activated to mount reactions against auto antigens such as the DNA from an auto cell
The mechanism of both the types can be summarized as follows:

 Antigen-antibody complexes are formed when antibodies bind to antigens.
 In case the complex is not cleared by normal process of phagocytosis, the immune complexes persist in the
circulation.
 The immune complexes subsequently deposit in tissues.
 The tissue deposited complexes activate the classical complement cascade.
 The complement fragments (e.g. C3a and C5a) that form during complement activation activate a variety of potent
mediators of inflammation causing an influx of neutrophils and monocytes to the site of deposition.
 The attracted neutrophils attempt to engulf the immune complexes. Since the complexes are deposited over the
tissues, the neutrophils do not succeed.
 Consequently, the neutrophils release a number of substances like prostaglandins, lysosomal enzymes, and free
oxygen radicals over the complexes causing damage to the tissues at the site of immune complex deposition.
 Additionally, the binding of the Fc region of antibody in the immune complex may bind to the Fc receptor on platelets
causing aggregation, blood clots and blockage of blood vessels leading to hemorrhages at the site.
Factors that causes deposition of immune complex and increase susceptibility to Type III hypersensitivity
reaction:
1. Persistent infection
– In persistent infection such as Malaria, large number of immune complexes are formed and deposited
in tissues
2. Complement deficiency
– Complement removes immune complexes from blood, but when complement system is deficient,
large amount of immune complexes circulates in blood and deposits in tissues.
3. Autoimmunity
– In autoimmune disease, large amount of immune complexes are formed and deposited in tissues.
4. Genetic defects
– In certain genetic defects, small and soluble immune complexes are formed that cannot be
phagocytosed.
CLINICAL MANIFESTATION AND EXAMPLES OF TYPE 3 HYPERSENSITIVITY
Types of Type 3 Hypersensitivity Reaction:

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A. Localized Type 3 Hypersensitivity Reaction

- It means it affects only one body part or organ
- It is a reaction occurring at the point of stimulation or injection of foreign substances
Arthus reaction
 Demonstrated by Maurice Arthus in 1903
 It is a hypersensitivity that occurs several hours to days following the intradermal injection of a vaccine into an animal
 It is marked by the formation of antigen- antibody complexes, accompanied by localized inflammation, pain, redness
and tissue destruction
 It occurs due to:
- Repeatedly exposed to Antigen
- Enough IgG is developed in the body
- Further Exposure reaction – local reaction occurs
o So, repeatedly exposed to antigen which develops enough IgG in the body, a localized reaction occurs with
further exposure to antigen
o For example: tetanus injection. If it is given, the next booster dose will be after 5 years, so if you will be
injected once again, there would be more antigen in your body so in this case there would be repeatedly
exposed to antigen which develops enough IgG in the body and a localized reaction occurs with further
exposure to antigen
 Occurs 3-8 hours
B. Generalized Type 3 Hypersensitivity

- Generalized or systemic it means affecting the entire body, rather than a single organ or body part
Serum sickness
 When a large amount of antigen enter blood stream and bind to antibody, circulating immune complexes forms
 If antigens are in significantly excess compared to antibody, the immune complexes formed are smaller and soluble
which are not phagocytosed by phagocytic cells leading to Type III hypersensitivity reaction
 The manifestation of serum sickness depends on the quantity of immune complex as well as overall site of deposition.
The site may vary but accumulation of complexes occurs at site of blood filtration
 During Serum sickness, the immune system falsely identifies a protein antiserum as a harmful substance (antigen).
The result is an immune response that attacks the antiserum. Immune system and the anti-serum combine to form
immune complexes, which cause the symptoms of serum sickness.
Other Causes
 Drugs containing protein moiety of other species (heterologous protein)
 Monoclonal and polyclonal antibodies prepared from rabbit, horse, or mouse serum (antithymocyte globulin, OKT-
3)
 Stings from insects, ticks, and mosquito bites
 Symptoms: fever, rash, and painful swollen joints
 Occurs 6-15 days; sometimes 3 weeks
- it occurs 6-15 days because when antigen is coming to the body it allows the antibodies to be developed and
development is extended to 6-15 days , and once the antibody is developed only then the antigen – antibody will
form and only then the complement activation will occur and systemic issue will occur.
Autoimmune diseases and other causes of Type 3 Hypersensitivity

 Autoimmune disease happens when the body’s natural defense system can’t tell the difference between your own cells
and foreign cells causing the body to mistakenly attack normal cells
 Type III Hypersensitivity reactions can also be triggered by autologous antigens as seen in several of the autoimmune
diseases:
- Systemic Lupus Erythematosus (SLE)
- Rheumatoid Arthritis (RA)
Systemic Lupus Erythematosus (SLE)

 Systemic- affecting multiple organs in the body
 Erythematosus - is reddening of the skin.
 SLE sometimes called Lupus
 Is an autoimmune disease with multisystem involvement. The condition is characterized by the presence of circulating
IgG and IgM autoantibodies to host tissue components
 The exact causes of SLE remain unclear however genetic, hormonal, and environment factors may contribute to the
development of the disease.
 Immune complex deposition involves multiple organs however the main damage occurs to the <joints , skin and
glomerular basement
Some of the criteria for the diagnosis of SLE:

 Malar (over the cheeks of the face) "butterfly" rash
 Discoid skin rash: patchy redness that can cause scarring
 Photosensitivity: skin rash in reaction to sunlight exposure
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 Mucus membrane ulcers: ulceration of the lining of the mouth, nose or throat
 Arthritis: 2 or more swollen, tender joints of the extremities
 Pleuritis/pericarditis: inflammation of the lining tissue around the heart or lungs, usually associated with chest pain
with breathing
 Kidney abnormalities: abnormal amounts of urine protein or cellular elements
 Brain irritation: manifested by seizures (convulsions) and/or psychosis
 Blood count abnormalities: low counts of white or red blood cells, or platelets
 Immunologic disorder: abnormal immune tests include anti-DNA or anti-Sm (Smith) antibodies, falsely positive
blood test for syphilis, anticardiolipin antibodies, lupus anticoagulant, or positive LE prep test
 Antinuclear antibody: positive ANA antibody testing
COMMON SELF-REACTIVE ANTIBODIES IN SLE:
1. Anti-Nuclear Antibodies (ANAs)

- For more than 60 years the detection of ANAs by indirect immune-fluorescence (IIF) staining has been
fundamental for the diagnosis of autoimmune diseases. The prevalence of ANAs among SLE is very high, but
they may also be detected in patients with other autoimmune, malignant or infectious diseases as well as healthy
controls
- The term ANA is not considered accurate because it embraces antibodies directed against components localized
in several cellular compartments including nuclear constituents, nuclear membrane, mitotic spindle apparatus,
cytosol, cytoplasmic organelles and cell membranes.
2. Anti-dsDNA Antibodies
- Anti-dsDNA antibodies are considered a diagnostic marker and one of the classification criteria for SLE
- They were first described in sera of SLE patients in 1957
- The anti-dsDNA positivity using different techniques not only results in a variation of associations with clinical
and biochemical manifestations of SLE but with other rheumatic and inflammatory conditions -in SLE, a histone
molecule of autologous origin might represent the carrier protein, activating non-tolerant T cells. Although the B
cell repertoire response is limited to the exposed determinants on the chromatin surface, just a few peptides may
be sufficient to activate TH cells with the potential to stimulate the whole array of chromatin-specific B cells,
explaining the comprehensive repertoire of chromatin-reactive IgG antibodies in SLE patients.
3. Anti-Nucleosome Antibodies Nucleosomes,

- Are complex structures in which histone octamers are surrounded by chromatin . This primary structure is
considered as the core nucleosome particle. However, nucleosomes in vivo are plastic and dynamic structures
which are associated with several other particles such as RNA, ribonucleoproteins, transcription factors and
enzymes
- The prevalence of these antibodies in sera of SLE patients is higher and is considered a more sensitive marker
compared to dsDNA antibodies -nucleosomes may play an important role in SLE through the induction of a T cell
mediated response by the hapten carrier-like system to raise several autoantibodies.
4. Anti-Sm Antibodies
- Sm antigens (named after their identification in the serum from a patient named Stephanie Smith) are a set of
seven core proteins (B, D1, D2, D3, E, F, G) forming a ring for small nuclear ribonucleoproteins (snRNP).
- The pathogenic role and the contribution of anti-Sm antibodies to the disease remain uncertain. However, they
are highly specific for SLE and represent one of the immunological diagnostic criteria for the disease
- Anti-Sm antibodies are found to react with neuroblastoma cell lines and they are also detected in the
cerebrospinal fluid of NPSLE patients and their levels are correlated with anti-NR2 antibodies.
- The sensitivity is low and they are only detected in 20% of Caucasian SLE patients and 30%–40% of African,
African-American and Asian patients.
5. Anti-RNP Antibodies
- The snRNP are RNA-protein complexes, abundant in the nucleus and involved in the nuclear processing of the
pre-mRNA along with other proteins constituting the spliceosome.
- The anti-RNP antibodies react with proteins (70 kDa, A, C) that are associated with the U1 RNA forming the
U1snRNP. The 70 kDa protein is one of the major determinants in the antibody response to U1-RNP: anti-70 kDa
antibodies are developed early in SLE pathogenesis and may contribute to the development of antibodies against
other proteins of the U1-RNP complex through the epitope spreading mechanism
- Anti-U1-RNP antibodies are detected in 20%–30% of SLE patients but they do not show a good specificity for
SLE since they are commonly found in mixed connective tissue disease (MCTD
6. Anti Ro/SSA and anti La/SSB Antibodies

- Anti-Ro and anti-La antibodies are found in 30%–40% and 10%–15% of SLE patients,
- No pathogenic mechanism has been attributed to anti-Ro and anti-La antibodies in SLE disease. However, women
with positive levels to these autoantibodies show a high risk to develop neonatal lupus in the fetus with congenital
heart block as the most serious symptom of the syndrome
7. Anti-Phospholipid Antibodies
- Anti-phospholipid antibodies (aPLs) are found in 30%–40% of SLE patients but they are not specific and can be
detected in other autoimmune diseases, infections and drug induced disorders, as well as in some healthy controls
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- Around half of SLE patients with aPLs develop antiphospholipid syndrome, an autoimmune disorder
characterized by recurrent arterial or venous thrombosis, pregnancy-related problems, thrombocytopenia,
hemolytic anemia and persistent elevated levels of aPLs
8. Anti-C1q Antibodies
- C1q deficiency has been described as a high risk factor to develop SLE but the genetically C1q deficiency in SLE
is very rare and the susceptibility risk of gene variants at the C1Q gene remains controversial]. Conversely, low
levels of C1q are typically associated with disease flares and with the appearance of anti-C1q antibodies which,
in turn, are found in 20%–50% of SLE patients
9. Anti-Ribosomal P (anti-P) Antibodies

- presence of anti-P antibodies is highly specific for SLE, the prevalence is low and variable (10%–20% to 40%)
and depends on the detection assay used Several groups have published the association of anti-ribosomal P
antibodies with some clinical features of SLE: neuropsychiatric lupus, LN and lupus hepatitis
- But the pathological mechanism attributed to anti-P antibodies remains uncertain. -The most frequent association
described has been between the SLE neuropsychiatric symptoms (psychosis, depression) and anti-P antibodies
levels
- The prevalence of anti-NMDAR antibodies is approximately 30% in SLE patients and their levels are highly
correlated with neuropsychiatric clinical manifestations
- Moreover, anti-NMDAR antibodies were found in lupus brain and in the cerebrospinal fluid, implying that the
break of the blood-brain barrier occurs in SLE even without a history of inflammatory events at the central
nervous system
TREATMENT AND MANAGEMENT of SLE

 SLE is treated based on the individual patient's disease condition. Hydroxychloroquine is essential for long-term
treatment in all SLE patients. Antimalarial, corticosteroids, nonbiologic DMARDS, nonsteroidal anti-inflammatory,
and biologic DMARDs are other medications used in the treatment of SLE.
Rheumatoid Arthritis (RA)

 Arth – refers to joints and
 Itis – means inflammation
 Rheumatoid – refers to Rheumatism which is broader refer to mucsoskeletal illness
 Rheumatoid arthritis, or RA, is an autoimmune and inflammatory disease, which means that your immune system
attacks healthy cells in your body by mistake, causing inflammation (painful swelling) in the affected parts of the
body.
 RA mainly attacks the joints, usually many joints at once
 Complement enhances tissue destruction in both diseases ( SLE, RA)
Autoantibodies in rheumatoid arthritis:

 Rheumatoid factors and anticitrullinated protein antibodies
- the two most remarkable autoantibodies in RA.
Rheumatoid Factor (RF)

 Was described by Waaler in 1940,1 and it was later found to be directed to the Fc region of IgG.
 Autoantigens targeted by a number of autoantibodies subsequently found in RA display a wide spectrum of cartilage
components, stress proteins, enzymes, nuclear proteins and citrullinated proteins, which demonstrates that RA is not
characterized by only one autoreactivity to a single autoantigen but by accumulated autoreactivities in both B and T
cells.
 In the antigen binding site of RF, the Fc-part of IgG, has fueled the hypothesis of infection origin for RA. RF was then
supposed to be an anti-idiotypic antibody to e.g. the protein G or protein A (both bacterial proteins) binding site.
 Basically Rheumatoid factors is an antibody which is like a dog chasing its tail and the result is not fun, that result
disease)
 However, this field of research has not led to new relevant insights in the pathogenesis of RA.
Anticitrullinated protein Antibodies

 Anti-citrullinated protein antibodies (ACPAs) are autoantibodies (antibodies to an individual's own proteins) that are
directed against peptides and proteins that are citrullinated. They are present in the majority of patients with
rheumatoid arthritis.
 The most specific autoimmunity known for rheumatoid arthritis (RA) is reflected by generation of anti-citrullinated
protein antibodies (ACPA).
 Presence of ACPA in established RA is associated with disease severity, while generation of ACPA at early
developmental phases of RA can have a strong predictive value for progressing to the full-blown disease. Hence,
development of ACPA may be of crucial importance to the pathogenesis of RA.
TREATMENT:
 Methotrexate as the first medication providers should consider when treating people with rheumatoid arthritis. In
head-to-head clinical trials, methotrexate was found to be equally or more effective, and have fewer side effects, than
other nonbiologic DMARDs
 Methotrexate is one of the most effective medications to treat RA because it will help ease symptoms like joint pain,
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fatigue, redness and swelling. It may also help prevent damage to your organs and joints
TESTING FOR TYPE 3 HYPERSENSITIVITY
In autoimmune diseases such as SLE and Ra, the presence of antinuclear antibodies can be detected by a variety of methods:
 Indirect Immunofluorescence
 Enzyme – linked immunosorbent assay
 Fluorescent microsphere multiplex immunoassays
 Fluorescent staining of tissue sections has also been used to determine deposition of immune complexes in the tissues-
the staining pattern seen and the particular tissue affected help to identify the disease and determine its severity.
 Rheumatoid factor can be detected by latex agglutination, nephelometry or other immunoassays
 Measuring complement levels is a method of evaluating immune complex- during periods of high disease activity,
complement levels in the serum may be decreased because of binding of some of the complement to the antigen-
antibody complexes, and the result should be interpreted in conjunction with other clinical findings.
TREATMENTS OF TYPE 3 HYPERSENSITIVITY
 Preventing further exposure to the antigen and the use of anti-inflammatory drugs
 Anti-inflammatory corticosteroid inhalers can also be used to diminish inflammation to allow lung lesions to heal
 Systemic corticosteroid treatment
 Oral or intravenous
● Type 4 hypersensitivity reaction was first described in 1890 by Robert Koch
- He observed that individuals infected with Mycobacterium tuberculosis (Mtb) developed a localized inflammatory
response after receiving intradermal injections of a filtrate from the organism
● Type IV hypersensitivity differs from the other three types of hypersensitivity in that sensitized T cells, rather than antibodies,
play the major role in its manifestations
● This reaction is also known as delayed hypersensitivity because symptoms peak between 48 to 72 hours after exposure to
antigen
- The reason why symptoms happen 48 to 72 hours or 2-3 days after exposure is because it takes time for the T cells to be
activated and differentiated, for the chemokines and cytokines to be secreted, and for the macrophages and other
leukocytes to be recruited to the site of antigen exposure
ANTIGENS OF TYPE 4 HYPERSENSITIVITY
1. Intracellular pathogens
- These microbes are those that escape elimination by immune mechanisms and cause prolonged infections
- Can be bacteria, fungi, parasites, or viruses
- Ex: Mycobacterium tuberculosis, Mycobacterium leprae, Pneumocystis carinii, Leishmania species, and herpes simplex virus
2. Contact antigens
- Extracellular antigens
- Those that come into direct contact with the skin
- They include plants such as poison ivy and poison oak, metals such as nickel salts, and components of hair dyes and
cosmetics
TWO STAGES OF TYPE 4 HYPERSENSITIVITY
1. Sensitization Stage
- Primary contact
- T cells are sensitized and memory T cells are produced
- 7-10 days (or 1-2 weeks)
2. Effector Stage
- Secondary contact
- Host Tissue damage
- 1-2 days
MECHANISM OF TYPE 4 HYPERSENSITIVITY

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● Cells play the major role in its manifestations unlike the first three types which are mediated by antibodies. Particularly these
cells are the T lymphocytes.
● When t cells mature, they will exhibit either CD4 or CD8 marker. The T cells bearing the CD4 receptor are termed helper, or
inducer cells, while the CD8-positive (CD8) population consists of cytotoxic or suppressor T Cells.
- CD4+ T cells are mediators of DELAYED TYPE HYPERSENSITIVITY. They release cytokines which are small proteins that can
stimulate or inhibit other cells such as macrophages and monocytes which can cause tissue damage
- CD8+ T cells are mediators of DIRECT CELL CYTOTOXICITY wherein the damage is done by the CD8+ T cells themselves and
no other types of cells are recruited or involved.
● Both CD8+ and CD4+ t cells are still considered as naive T cells since they are not yet exposed to antigens. But once they bind
with the antigen, they will be activated and differentiated.
● If peptides fragment derived from antigens are presented in complex with MHC class 2 molecules, CD4+ T cells are activated
● If antigens are presented in complex with MHC class 1 molecules, CD8+ T cells are activated
Mechanism of CD4+ T cell hypersensitivity reaction

● Contact dermatitis – it is a type of skin allergy to plants like the poison ivy
● The poison ivy contains the allergen called UROSHIOL (a small molecule that binds and alters the proteins in the skin)
● Once the body respond to the molecule (urushiol), this marks the beginning of the sensitization stage.
● Urushiol is small enough to quickly make its way through the epidermis and to the dermis, which is where it will combine with
small proteins present in the skin
- Uroshiol is considered haptens since it needs larger molecules like proteins present in the skin to initiate an immune
response
- Once these haptens bind to the proteins, the immune system will recognize the changes in the proteins as foreign
● These immunogens are then recognized and processed by antigen presenting cells (APCs) and take it to the nearest lymph node
where it presents the antigen on its surface using a MHC class 2 molecule (a serving platter for CD4+ T cells)
● If a Th cell recognizes the antigen, it binds to the MHC class 2 molecule using its T cell receptor (TCR)
● The CD4+ or T helper cell will express a CD28 protein which will bind to the B7 protein on the surface of the dendritic cell
● Once it binds to the TCR and the CD28 protein, the dendritic cell releases IL-12 which causes the naïve T helper cell to mature
and differentiate into Th1 cells
● The APC could also produce cytokines like IL-1, IL-6, along with TGF-β that causes the CD4+ T cell to differentiate into Th17
subset
● The mature T cells (Th1 and Th17) form an immunological memory of the antigens that lead to their activation
● The memory T cells stay in the circulation and upon re-exposure to the allergens, these memory T cells will quickly recognize
these antigens displayed by APCs and respond
● The effector stage begins when there is a subsequent exposure to antigen (re-exposure)
● Upon re-exposure to the antigen, the antigens are again picked up by the APC and this time presented to the Th1 cell
● On recognition of the antigen, the Th1 cells secretes many cytokines:
- IL-2 – helps both Th1 and other T cells in the area to proliferate
- IFN-γ – activates phagocytes like macrophages and creates more Th1 cells
o The activated macrophages have better antigen presenting abilities, increased MHC II expressions, and improved
phagocytosing capabilities
o The activated macrophages also produced TNF-α, IL-6, and IL-1 which cause more leukocytes to leak out of the nearby
blood vessels
o It also secretes lysosomal enzymes, complement components, and reactive oxygen species into the exposed area to
help remove the offending agent
● Th17 cells mainly secretes cytokines IL-17 and IL-22
- Their main job is to recruit more inflammatory cells like neutrophils and monocytes to the site of allergy and cause similar
inflammatory actions like the macrophages.
● Many cytokines and chemokines are secreted by Th1 and Th17 cells which cause the recruitment of many different
inflammatory cells in the site of allergy. As a result, there is an infiltration of large number of inflammatory cells in the dermis as
well in epidermis (site of allergy)
- Poison ivy mainly affects the skin and causes contact dermatitis. Dermatitis is the inflammation of the skin
● These inflammatory cells destroy the allergens by releasing various enzymes and also by phagocytosis. These enzymes can
damage our own skin cells
● As a result, local cell damage, inflammation, and edema will occur which are manifested as redness, itching, increase
temperature, and development of vesicular eruption at the site of contact
Mechanism of CD8+ T cell hypersensitivity reaction

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● CD8+ T cells can target antigen when they’re presented on MHC class I molecules, which are present on all nucleated cells
● Example of intracellular antigens: viral, tumor, and certain parasitic antigens that are synthesized within the cell
● MHC class I molecules present antigens from inside the cell, so this process is particularly important when cells become infected
with viruses or mutated like with cancer.
● The CD8+ T cells does not activate macrophages and neutrophils, instead they add themselves and destroy the antigen bearing
cells
● An effector cytotoxic T cell specific to that antigen would use its TCR to bind to the MHC class I molecule, which would cause it
to release perforin and granzymes
● Perforins would perforate the target cell by forming pores. These pores allow the granzymes to enter the cell.
● Granzymes are proteases that induce apoptosis (program cell death) once inside the target cell
CLINICAL MANIFESTATION AND EXAMPLES OF TYPE 4 HYPERSENSITIVITY
CD4+ T cell Reactions:
Contact Dermatitis
● Reactions are usually caused by low-molecular-weight compounds that touch the skin.
● The most common causes include poison ivy, poison oak, and poison sumac, which release the chemical urushiol in the plant
sap and on the leaves
- Urushiol is a low-molecular-weight compound therefore it is considered as haptens that cannot induce immune response
by themselves
- They bind to larger molecules (proteins) to be recognized as immunogens which can induce an immune response
- Prior the binding with larger molecules, these molecules are still considered as incomplete antigens since epitopes are only
present in their surfaces but once they bind to larger molecules (carrier) they will become complete antigens (can induce
an immune response)
● It can also happen in response to wearing nickel (often found in earrings and necklaces); rubbers; contact to formaldehyde;
hair dyes and fabric finishes; cosmetics; and medications applied to the skin (topical anesthetics, antiseptics, and antibiotics)
● Latex sensitization – contact dermatitis to many health care workers
- The use of latex gloves
- Any product containing latex when being exposed to the skin can induce an allergic response
● Tuberculin skin test (or PPD) is also an example of contact dermatitis since it also causes allergy in the skin
- It is use for testing if a patient has been infected with Mycobacterium tuberculosis
- A protein components of the bacteria Mycobacterium tuberculosis is injected in the skin
- If there has been a previous exposure to TB, it develops into type IV reaction where TB-specific Th1 cells will migrate to the
injection site and created an inflammatory response that results in the thickening or hardening of the skin which is also
called induration.
● Contact dermatitis produces a skin eruption characterized by erythema, swelling, and the formation of papules that appears
from 6 hours to several days after the exposure
● The dermatitis is first limited to skin sites exposed to the antigen, but then it spreads to adjoining areas.
● Dermatitis can last for 3 to 4 weeks after the antigen has been removed
Hypersensitivity Pneumonitis
● Allergic disease of the lung parenchyma characterized by inflammation of the alveoli and interstitial spaces.
● Mediated predominantly by sensitized T lymphocytes that respond to inhaled allergens
● It is caused by chronic inhalation of a wide variety of antigens and is most often seen in individuals who are engaged in work or
hobbies involving exposure to the implicated antigen
● Example diseases: farmer’s lung, bird breeder’s lung disease, and humidifier or air conditioner lung disease
● The reaction is most likely caused by microorganisms, especially bacterial and fungal spores
● Symptoms: dry cough, shortness of breath, fever, chills, weight loss, and general malaise, which may begin 6 to 8 hours after
exposure to a high dose of the offending antigen
Granulomatous diseases
● Include tuberculosis, sarcoidosis, leprosy, and cat-scratch disease
● This happens when a persisting antigens cause a continuous loop of Th1 cells activating macrophages in the site of reaction
● These macrophages cannot remove the target antigen completely
● For example, the walls of Mycobacterium tuberculosis contains mycolic acid which the macrophages phagocytose but cannot kill
them
● Since they cannot kill the bacteria, the macrophages surround and try to isolate it from the surroundings
● Constant activation of macrophages by IFN-γ causes macrophages to transform into epitheloid cells (resembles epithelial cells)
● The epitheloid cells are also surrounded by lymphocytes and fibrosis
● This mass is called a granuloma that causes granulomatous inflammation
CD8+ T cell Reactions:

● Type 1 diabetes – the cytotoxic t cells target insulin-secreting beta cells of the pancreas
● Rheumatoid arthritis – the cytotoxic t cells Target joint tissue
● Hashimoto disease – attack thyroid hormone producing follicular cells
● Graft rejection – target graft cells
TESTING TYPE 4 HYPERSENSITIVITY
SKIN TESTING FOR DELAYED HYPERSENSITIVITY

 Also known as the Mantoux tuberculin skin test (TST)
 Considered the gold standard in testing for contact dermatitis
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TST MECHANISM
 It is based on T-cell-mediated memory response
 When antigen is injected intradermally or applied to the surface of the skin, previously sensitized
individuals develop a reaction at the application site
 Due to the infiltration of T lymphocytes and macrophages into the area
 Blood vessels in the vicinity become lined with mononuclear cells
 Peak: 72 hours after exposure
TST APPLICATIONS
 Has been used to determine allergen sensitivity in contact dermatitis
 To asses exposure to Mycobacterium tuberculosis
 To evaluate competency of cell-mediated immune responses in patients with immune deficiency diseases
SKIN TESTING FOR EXPOSURE TO TUBERCULOSIS

 Based in the principle that soluble antigens from Mycobacterium tuberculosis induce a reaction in people
who currently have tuberculosis or have been exposed to M. tuberculosis in the past
 TST uses an M. tuberculosis antigen extract prepared from a purified filtrate of the organism’s cell wall,
called a purified protein derivative (PPD)
 Routinely performed by the Mantoux method
SKIN TESTING FOR DETERMINING CELL-MEDIATED IMMUNE RESPONSE

 TST is used to determine whether cell-mediated arm of the immune system is functioning properly in
individuals suspected of having immunodeficiency disorders
 Antigens typically used for testing:
- Candida albicans
- Tetanus toxoid
- Streptococcus bacteria
- Fungal antigens: trichophyton and histoplasmin
 Injected intradermally by the Mantoux method
HOW IS THE TST ADMINISTERED?

 This test must be done when the patient is free of symptoms or at least has a clear test site
 The TST is performed by injecting 0.1 mL of tuberculin purified protein derivative (PPD) into the inner
surface of the forearm/back
 The injection should be made with a tuberculin syringe, with the needle bevel facing upward
 The TST is an intradermal injection
 When placed correctly, the injection should produce a pale evaluation of the skin (a wheal) 6 to 10 mm in
diameter
HOW IS THE TST READ?

 Should be read between 48 to 72 hours after administration of test for the presence of hardened, raised
area called induration
 Presence of indurations is considered a positive test
 Skin Testing for exposure in TB:
- 15 mm or more: positive in individuals with no risk factors
- 10 mm or more: positive in recent immigrants of high prevalence countries, IV drug users, HCWs &
other high-risk facilities, persons with clinical conditions and children younger than 5 years of age
- 5 mm or more: positive in persons who have HIV infection or other immunosuppression, features on a
chest x-ray consistent with TB, or recent contact with TB patients
 Skin Testing for determining cell-mediated immune response:
- Normal: develop a positive skin reaction to at least one of the antigens tested
- Deficient: display anergy, absence of positive reactions for all of the common antigens tested
 Final evaluation is conducted at 96 to 120 hours
 False-Positive Reactions:
- Previous TB vaccination with the bacille Calmette-Guerin (BCG) vaccine
- Infection with non-tuberculosis mycobacteria (mycobacteria other than M. tuberculosis)
- Incorrect measurement or interpretation of reaction
- Incorrect antigen used
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NOTE: A TB blood test is the preferred method of testing for people who have received the BCG vaccine in
order to prevent false-positive reactions. TB blood tests are also called interferon-gamma release assays or
IGRAs.
 False-Negative Reactions:
- Anergy
- Recent TB infection (within the past 8 to 10 weeks)
- Very young age (younger than 6 months)
- Recent live-virus measles or smallpox vaccination
- Incorrect method of giving the TST
- Incorrect measuring or interpretation of TST reaction
● There is no cure for these diseases. The treatment only aims at symptom control
● Strategies to avoid Type Iv Hypersensitivity reactions include:
- Avoiding antigen exposure
o In the case of contact dermatitis, a patient needs to avoid contact with the offending allergens such as those plants,
the poison ivy, poison oak; metals or nickel salts present in necklaces and earrings; hair dyes and cosmetics; latex
gloves or any latex products
- Administration of anti-inflammatory drugs or corticosteroids
o Contact dermatitis: If the area of allergy is small and localized, a topical steroid may be used for treatment. But if it is
systemic, corticosteroids may be administered
o These systemic corticosteroid therapy is also used as treatment for Hypersensitivity Pneumonitis.
- Administration of other drugs that alter the body’s immune system including interferon, cyclophosphamide, cyclosporine
etc.
o Example: TNF-α monoclonal antibodies and recombinant interferon-β
SUMMARY
 Hypersensitivity is an exaggerated immune response to antigens that are usually not harmful. It results in cell
destruction and tissue injury.
 Gell and Coombs devised a system for classifying hypersensitivity reactions into four types based on the immuno-
logic mechanism involved and the nature of the triggering antigen.
 Hypersensitivity types I, II, and III are antibody-mediated. Because they occur within minutes to hours after exposure
to antigen, they are referred to as immediate reactions. Type IV hypersensitivity is a cell-mediated response involving
T lymphocytes. Because the clinical manifestations do not appear until 24 to 72 hours after contact with the antigen,
the type IV response is also referred to as delayed hypersensitivity.
 Type I hypersensitivity is a Th2 polarized immune response that involves production of IgE antibody to the inducing
antigen or allergen. In the sensitization phase of this response, IgE antibody binds to high-affinity FcεRI receptors on
mast cells and basophils. In the activation phase, the receptors become cross-linked when the allergen binds to
adjacent IgE molecules. This cross-linking stimulates the cells to degranulate and release preformed and newly
synthesized chemical mediators that cause an inflammatory response. The reaction occurs very quickly, within
minutes of exposure to the inducing antigen. Cytokines produced during the response can cause a late- phase response
of prolonged inflammation.
 Preformed mediators that are released from mast cells and basophils include histamine, eosinophil chemotactic factor
of anaphylaxis, neutrophil chemotactic factor, and proteolytic enzymes such as tryptase. These factors cause
contraction of smooth muscle in the bronchioles, blood vessels, and intestines; increased capillary permeability;
chemotaxis of eosinophils and neutrophils; and decreased blood coagulability. Newly synthesized mediators such as
prostaglandins, leukotrienes, and PAF potentiate the effects of histamine and other preformed mediators.
 Clinical manifestations of type I hypersensitivity include localized wheal-and-flare skin reactions (urticaria or hives);
rhinitis; allergic asthma; and systemic anaphylaxis, which can be life-threatening.
 Susceptibility to allergies is based on a combination of genetic factors and environmental influences. Genes affect
different aspects of the immune response that contribute to the pathogenesis of type I hypersensitivity. Some genes
affect anatomical structures. Exposure to infectious organisms appears to play a key role in the development of
allergic disease. Stress, variations in physical factors such as temperature, and contact with environmental pollutants
can intensify clinical manifestations of allergy in susceptible individuals.
 Allergies can be treated with drugs such as antihistamines, decongestants, and corticosteroids. The monoclonal anti-
IgE antibody omalizumab has been used to block the binding of IgE to mast cells and basophils in patients with
moderate to severe asthma.
 Allergen immunotherapy (AIT) can be administered to patients for whom drug therapy and environmental control
measures are not successful. The goal of AIT is to induce immune tolerance by administering gradually increasing
doses of the allergen over time.
 The preferred method of screening for allergies is an in vivo skin prick test, in which very small amounts of potential
allergens are injected under the skin. A positive test produces a wheal-and-flare reaction within 20 minutes.
 In patients unable to tolerate skin testing, in vitro testing by noncompetitive solid-phase immunoassays for allergen-
specific IgE can be performed. In these assays, patient serum is incubated with a solid phase to which a specific
allergen has been attached. Binding is detected with an enzyme-labeled anti-human IgE antibody and a colorimetric,
fluorescent, or chemiluminescent substrate.
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 Solid-phase immunoassays for total serum IgE can be used to monitor patients undergoing treatment with AIT or
omalizumab or to detect patients with certain diseases characterized by elevated IgE levels. The principle of these
tests is the same as that for allergen-specific IgE tests except that anti-IgE, rather than allergen, is attached to the solid
phase.
 Type II hypersensitivity involves production of IgG or IgM antibodies to antigens on the surface of host cells. These
antibodies can destroy the cells through complement-mediated cytolysis, opsonization and phagocytosis, or antibody-
dependent cellular cytotoxicity (ADCC). In other cases, binding of the antibody to the cell surface antigen can result
in dysfunction or overstimulation of the cell.
 Examples of type II reactions that involve cell damage include autoimmune hemolytic anemia, transfusion reactions,
and hemolytic disease of the newborn (HDN). Myasthenia gravis is an example of a type II disorder in which the
antibody blocks binding of a ligand to cell receptors, causing dysfunction of the cells. In contrast, in Graves’ disease
the antibody produced stimulates cells after binding to cell receptors.
 The direct antiglobulin test (DAT) is used to screen for transfusion reactions, autoimmune hemolytic anemia, and
HDN. In this test, washed patient RBCs are combined with anti- human globulin and observed for agglutination,
indicating the presence of IgG or complement components on the cells.
 Cold agglutinin antibodies bind to RBCs at temperatures below 30°C and cause micro-occlusions of small blood
vessels or destruction of the RBCs, mainly through opsonization and extravascular clearance by macrophages in the
liver. Production of cold agglutinins may be from unknown causes or may be associated with certain infections or B
cell/plasma cell lymphoproliferative disorders. Cold agglutinin titers can be determined by incubating patient serum
with a dilute suspension of human type O RBCs overnight at 4°C and observing for agglutination.
 Type III hypersensitivity involves formation of IgG or IgM antibody that reacts with soluble antigen under conditions
of slight antigen excess to form small complexes that precipitate in the tissues. These complexes activate complement,
resulting in migration of neutrophils to the site with subsequent release of lysosomal enzymes that produce damage to
the surrounding tissues.
 The Arthus reaction, characterized by deposition of antigen–antibody complexes in the blood vessels of the skin, is a
classic example of a type III reaction. Other examples include serum sickness and autoimmune diseases such as SLE
and RA.
 Type IV hypersensitivity is a cell-mediated mechanism that involves the activation of Th1 cells to release cytokines.
As a result, macrophages and other immune cells are recruited to the area, where they induce an inflammatory
reaction. Cytotoxic T cells may also cause damage to the target cells involved.
 Contact dermatitis is an example of a type IV hypersensitivity reaction. It results from exposure to chemicals released
by plants such as poison ivy and poison oak, metals such as nickel, or components of hair dyes and cosmetics that act
as haptens when bound to self-proteins. Hypersensitivity pneumonitis is a type IV hypersensitivity response that
results mainly from occupational exposure to inhaled antigens.
 Skin testing is used to detect the type IV hypersensitivity responses in contact dermatitis and tuberculin (PPD) testing.
It is also used to test for functional cell-mediated immunity to common antigens in patients suspected of having
immunodeficiency diseases. Positive test results appear in 48 to 72 hours and indicate sensitization to the antigen(s)
used in the test.
 All four types of hypersensitivity represent defense mechanisms that stimulate an inflammatory response to cope with
and react to an antigen that is seen as foreign. In many cases, the antigen is not harmful, but the response to it results
in tissue damage.
REPORTERS:
Zipagan, Gianne D.
Ramos, Ia Micah N.
Pascua, Kate Dhanielle
Cadiente, Alyssa Jade R.
Honorio, Gregwin M.
Dunbar, Hannie Grace M.
Tan, Allan Carl Wilfred C.
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IMMUNOLOGY AND

TRANSPLANT IMMUNOLOGY SEROLOGY (IS)

TRANSPLANT IMMUNOLOGY intestine) transplants were performed

NOTE: HLA proteins are found on the Major Histocompatibility Complex

The human leukocyte antigen (HLA)

Minor Histocompatibility Antigens

HLA match. Up to one third of

NOTE: ABO blood group incompatibility

Clinical Transplantation Killer Immunoglobulin-Like Receptors

number and type on any

Proteins described post-transplantation Allograft (homograft)

 The HLA (human leukocyte

Effector responses against transplanted Such antibodies have been

IN HUMANS, GVHD MAY

NOTES: 4. Monoclonal antibodies

when we say amplify, in layman’s

of either molecular or the genetic

SANGER DIDEOXY CHAIN

 The dideoxy terminator are Provide varying levels of resolutions

These antibodies can develop in

calculated PRA (cPRA)

flow cytometry using an FITC labeled

flow cytometric crossmatch

Welsh, K. (1999). Molecular typing for the

Turgeon, M. L. (2013). Immunology &

(n.d.). Retrieved from

The molecular defects, signs, and symptoms

[BSMT 2B- 2020-2021- Group 4] Page 1

DEFECTS OF PHAGOCYTIC CELLS DEFECTS OF T LYMPHOCYTES

● Patients with defects in B lymphocytes can

[BSMT 2B- 2020-2021- Group 4] Page 2

B- and T-Cell Deficiencies

[BSMT 2B- 2020-2021- Group 4] Page 3

Immunodeficiency The PIDs can affect one or more parts of the

[BSMT 2B- 2020-2021- Group 4] Page 4

 result in pyogenic (i.e., pus-forming) in neutrophil function are usually reflected

[BSMT 2B- 2020-2021- Group 4] Page 5

[BSMT 2B- 2020-2021- Group 4] Page 6

 This category encompasses conditions in in response to environmental stimuli.

[BSMT 2B- 2020-2021- Group 4] Page 7

[BSMT 2B- 2020-2021- Group 4] Page 8

 Three major types of cellular defects have normal range.

This category contains diseases in which there are

[BSMT 2B- 2020-2021- Group 4] Page 10

effects on immunoglobulin levels as well. adapter molecules, downstream

[BSMT 2B- 2020-2021- Group 4] Page 11

This category differs from Category 1 in that

[BSMT 2B- 2020-2021- Group 4] Page 12

defect, and located on the X chromosome,

[BSMT 2B- 2020-2021- Group 4] Page 13

hypoparathyroidism) or manifectations of autosomal recessive syndrome

 Apparently essential to the recombination

[BSMT 2B- 2020-2021- Group 4] Page 14

 Allogeneic bone Marrow transplantation. Immunodeficiency with hypopigmentation (

 Normal numbers of T or B cells but with

The Autoimmune Lymphoproliferative

 May involved in apoptosis. Defective apoptosis

_______ Play a crucial role in the

[BSMT 2B- 2020-2021- Group 4] Page 15

 Neutrophils must adhere to vascular

 Defect affecting each of these steps can

Chronic Granulomatous Disease

It was historically diagnosed by measuring