Diagnostic Immunopathology….

Immunodeficiency Disorders
 What is Immunodeficiency?
Under-reaction to antigen

• A failing of one or more of the body’s defensive

mechanisms resulting in morbidity or mortality.
• Any part of the immune system can be deficient –
cells, proteins, signalling mechanisms…….
• The body is susceptible to infection by organisms that
meet with little or no resistance.
• Or, in certain cases, other homeostatic systems in the
body will be disrupted by the defect.
• Severity is variable.
• Immunodeficiency may be Primary or Secondary.
 Under-reaction to antigen
Immunodeficiencies

• occur when some part of the immune system

is defective or missing
– T cells, B cells, phagocytes, or complement

• these deficiencies are grouped as:

– primary: inherited or congenital
• severe combined immunodeficiency
(SCID)
– secondary: acquired
• HIV infection David Vetter 1972-1984,
the original bubble boy
Primary Immunodeficiency:

• Arises without a pre-existing

causative disorder.

• Usually has a genetic basis.

Secondary Immunodeficiency:

• Arises because of another pre-existing

pathology or intervention, e.g.
– Infection
– Renal failure, or protein losing enteropathy
– Leukaemia or Lymphoma
– Myeloma
– Extremes of age
– Certain Drug Therapies
Immunodeficiency Disorders
• Most immunodeficiency disorders are
acquired (most common acquired disorder
is AIDS)
• Approximately
• 50% are B cell disorders;
• 40% T cell disorders; and
• 5% each for phagocytic and complement
problems
• Most cases are in children less than 15
years of age (80% male)
• Phagocyte deficiencies:
– Cyclic neutropenia, oscillation of neutrophil numbers.
– Chronic Granulomatous disease,
– Specific granule defects, defective killing.
– Adhesion molecule defects, affects chemotaxis.

• Antibody deficiencies:
– X-linked agammaglobulinaemia
– Selective Ig deficiency, differentiation failure leading to
decrease in one or more of the IgG subclasses or IgA
subclasses.
– Common variable immunodeficiency (CVID), a common but poorly
defined collection of syndromes with reduced IgG, and
IgA/IgM.
• T cell deficiencies:
– DiGeorge’s Syndrome, gene defects affecting thymus
development and hence T cells. Now known as CATCH-22
syndromes.
– Wiskott-Aldrich, progressive reduction in T cells, also low
platelets and low IgM.

• Complement deficiencies:
– Deficiency of individual components, e.g. C3, C5 – C9, or
inhibitors Factor H, Factor I.
– Hereditary Angioedema (C1-INH deficiency).
Primary Immunodeficiency:

Phagocyte-specific Disorder
 Chronic Granulomatous
? Disease (CGD) ?
What is CGD?
• A group of diseases in which there is a defect in a
major killing mechanism of phagocytes.
• The diseases are rare, inherited and result from

? genetic abnormalities in key enzymes of the

‘oxidative burst’.
• CGD is the most significant of the phagocyte
defects.
• Patients are vulnerable to specific bacterial and
fungal infections.
?
 Clinical findings
• Recurrent severe bacterial and fungal infections
occurring at sites acting as environmental barriers:
– Skin -Lymph nodes
– Liver or spleen -Lungs
– Gastrointestinal tract
• Infecting organisms are specifically ‘catalase
positive’ which means the faulty phagocyte cannot
use the organism’s endogenously produced hydrogen
peroxide to kill them.
• Patients are no more susceptible to viral infection
than anyone else.
• Organisms include: Staph. Aureus, Salmonella,
Pseudomonas, Aspergillus, Candida…..and unusual
organisms such as Serratia marcescens
•
• Patients are usually children, although milder forms
may present in adulthood

• Symptoms may include boils, diarrhoea, gum disease,

fever, abscesses, lymphadenopathy, pneumonia, GI or
urinary obstruction.

• Chronic inflammation at various sites – e.g. gingivitis,

lymphadenopathy, granuloma.

• Delayed growth and malabsorption in children.

 Laboratory Diagnosis
• Patients often
have an elevated • Nitroblue tetrazolium test
white cell count (NBT) where NBT (colourless)
(neutrophil is reduced to formazan (blue)
by the phox enzymes.
leucocytosis).
Slide or tube assay – observing the
presence of blue inclusions in
phagocytes.
• Neutrophils are
morphologically
normal. • Dihydro-rhodamine (DHR)
reduction assay – fluorescent
rhodamine (reduced DHR) is
• Chemotaxis and detected by flow cytometry.
endocytosis are Flourescence in cells is measured and
normal. flourescing cells are counted.
 When to suspect CGD?

• In a patient (particularly a male child):

• with recurrent infection with catalase
positive organisms
• Infection with rare organisms
• Infection in unusual sites e.g. St.
aureus liver abscesses
 Therapy and Management
• Prompt aggressive treatment of
infection with antibiotics.
• Antibiotic prophylaxis.
• Trials of Gamma IFN – may reduce
number and severity of episodes.
• Steroids and NSAIDS sometimes
used to treat granulomas.
• Tissue biopsy/drainage – may need
hospitalisation.
• Individual susceptibility varies but
1 serious infection can be expected
every 3-4 years in X-CGD cases
even with prophylaxis.
Primary Immunodeficiency:

Disorder of the Complement

System

Hereditary Angioedema (HAE)

 Complement Disorders

• C1 esterase inhibitor deficiency (hereditary angioedema)

– Autosomal dominant disease resulting in excessive
release of C2-derived kinins that increase vessel
permeability resulting in edema
• See involvement of the face and oropharynx
sometimes with obstruction of the airway; also GI
• Laboratory tests show a decreased level of C4
(best screen) and C2. A low level of C1 esterase
inhibitor is the confirmatory test
• Treatment with synthetic androgens which increase
the synthesis of the inhibitor
? What happens in HAE? ?
• A deficiency of C1INH due to genetic mutations
of the C1INH gene on chromosome 11p11-q21.

• The loss of control of the complement and

contact systems results in severe subcutaneous
and submucosal oedema due to fluid leakage into
the extravascular space.

• Patients are also susceptible to immune complex

disease and SLE (failure to remove IC’s).
– Secondary or Acquired forms (AAE) occur in
some LPD’s or in association with C1INH

? autoantibodies.
?
 Different variants exist:
• Variant I • Variant II

• 80-85% of cases • 10-15% of cases

• Low levels of normal • Normal or increased levels of
C1INH are found (10- abnormal C1INH are found
30% of normal) • Autosomal dominant, genetic
• C1INH present is mutations are usually single
produced by the normal ‘point mutations’
allele
• Autosomal dominant, • Variant III
genetic mutations often • Not associated with C1INH
‘missense mutations’ deficiency (X-linked)

No one with homozygous HAE has ever been observed.

 Clinical findings

• Occurs in childhood or adolescence.

• Extreme swelling of tissues – oedema.

• Often in hands, face, feet, airways.

• Pain and swelling of abdomen, nausea and vomiting – often

mistaken for acute abdomen.

• Swelling of larynx – may cause fatal asphyxiation.

• Tingling or tightness may precede attacks.

• Oedema lasts usually 24-72 hours.

• Episodes may be as frequent as weekly or as rarely as once

a year or less.
 Triggers………
• Minor trauma
• Surgery
• Anxiety/stress
• Mild illness – colds or ‘flu
• Dental procedures
• Hormones – pregnancy or OCP
• Physical activity – typing, writing, gardening
etc….
 Laboratory Diagnosis

• Firstly - measure C4 levels.

If low:
• Measure C1INH level (single
radial-immunodiffusion)
and function (ELISA).
• Low C1INH with normal C1, C3, C1q (Type I HAE)
• Normal or raised levels of dysfunctional C1INH with
normal C1, C3, C1q (Type 2 HAE)
Primary Immunodeficiency:
Lymphocyte-specific disorder
affecting antibody production.

X-Linked Agammaglobulinaemia
- XLA (Bruton’s Disease)
 Clinical findings
• LITTLE BOYS WITH BIG INFECTIONS!

• Symptoms appear at 6-9 months of age (after loss of

maternal Ig) or as late as 3-5 years of age.

• Sites of infection: mucous membranes, ear (otitis

media), lungs (bronchitis/pneumonia), blood (sepsis), gut
(Giardia, or enterovirus), skin, eyes, meningitis.

• Also seen: joint problems, kidney

problems, neutropenia, malignancy in
older patients.
• Typical organisms involved include: Haemophilus
Influenzae, Streptococcus Pneumoniae.

• Affected boys have little or no tonsils or palpable

lymph nodes.

• Growth retardation may be present.

• There may be no family history of XLA.

• If diagnosis has been delayed then permanent lung

damage (bronchiectasis), or hearing loss due to
repeated infection may be present.
 Significant Laboratory Findings

Serum protein electrophoresis

• Microbiological evidence of
early-onset, severe, recurrent
infection.

• Profound reduction in
circulating B lymphocytes.

• Hypogammaglobulinaemia
(decreased immunoglobulin
levels) – absent in severe
cases.

• For confirmed diagnosis –

mutation in the Btk gene.
 Counting lymphocytes by
Flow Cytometry
488 nm

CD19-PE

CD3-FITC

CD45-Per CP
Lymphocyte subsets by Flow
Cytometry
 Therapy Options & Management
• Intravenous
Immunoglobulin (IvIg).

• Prompt treatment of any

infection and use of
prophylactic antibiotics.

• Avoidance of live
vaccines (Polio), including
siblings.
• Education of patient and
family.

• Genetic counselling.
• Prenatal testing.
 Prognosis

• No treatment:
– Death at an early age.

• Treatment but with chronic lung disease:

– May have a shortened life span.

• Early diagnosis and treatment:

– Normal active life.
 AIDS

• Acquired Immunodeficiency • caused by the human immunodeficiency

syndrome is the most virus (HIV - 1984) and is spread by
common acquired contact with body fluids
immunodeficiency in the E.U.,
U.S., first identified in 1981 • infects CD4+ (helper) T cells, which
– HIV 1 is the most common decrease in number
isolate in the E.U., U.S.,
Canada; HIV 2 is primarily • decreased numbers of CD4+ T cells lead
isolated in West and Central
Africa-transmitted to increased susceptibility to
heterosexually opportunistic infections.
– AIDS is the most common
cause of death in men and • treatments include drugs that inhibit
women between 25-44 years RT-ase, the activity of HIV proteins,
of age
thereby preventing production of the
virus
 AIDS
Modes of Transmission
• Subsets of individuals that become HIV positive in decreasing
order of frequency
– Homosexual or bisexual men
– IV drug abusers
– Heterosexual contacts
– Individuals receiving blood or blood products (includes
hemophiliacs)
• Men account for approximately 88% of all people who are HIV
positive (women-IV drug abusers- represent the remainder)
• Adults:
– Intimate sexual contact is the most common mode of transmission,
usually by anal intercourse between homosexuals or by vaginal
intercourse in heterosexuals
– Inoculation of the virus through blood or blood products is the second
most common mode of transmission with the majority of cases related
to the sharing of needles between intravenous drug abusers and less
commonly by receiving a blood transfusion (1:40.000 to 1:200.000 risk)
– Health care workers most commonly contract the disease from
accidental needle sticks where the risk for becoming HIV positive
is 0.3%
 AIDS
Modes of Transmission
• Children
– Aids is contracted in greater than 90% of cases from an infected mother
who is usually an intravenous drug user
– Transplacental (vertical) transmission accounts for approximately 30-50%
of cases, while perinatal factors associated with blood contamination
during delivery and breast feeding (most common perinatal factor)
represent the remainder of cases
– Pregnant women who have CD4 helper T cell counts less than 400
cells/uL or who are positive for the viral p24 antigen (a marker of
disease activity) have an increased risk for transmitting HIV to their
baby.
– Treatment of asymptomatic pregnant women with AZT has reduced the
rate of infants developing AIDS from 13-40% to less than 10%
• All newborns of HIV positive women are HIV Ab positive due to
transplacental passage of IgG antibodies; therefore, documentation
of HIV infection in infants is often difficult during the first 2
months
– Tests in this setting that are useful include the isolation of the
virus in peripheral blood monocytes, the detection of viral RNA
using PCR, and assays for the p24 antigen in the serum
AIDS
• AIDS virus attacks and destroys
CD4 T helper cells by first
attaching to the CD4 molecule on
the surface membrane of the
lymphocyte with the gp120
envelope protein

• A co-receptor must also be

present with the CD4 molecule for
fusion and entry of T cell tropic
strains of HIV-1.

• The co-receptor is CXCR4 on T

cells and  chemokine receptor
CCR5 on macrophages

• Macrophages are the primary

reservoir for the virus and are
the vehicle for carrying the
virus into the CNS
 AIDS
• Following binding, fusion with
the host cell membrane occurs
via the gp41 molecule.

• The HIV genomic RNA is

uncoated and internalized.
Reverse transcriptase catalyzes
the reverse transcription of
viral RNA into double-stranded
DNA.

• The virally encoded enzyme,

integrase, causes the viral DNA
to be integrated into the host
cell DNA
AIDS
• Cells with CD4 molecules on their
surface in which the virus can
replicate without killing the cell
are monocytes, tissue
macrophages, dendritic cells in
the skin and lymph nodes, and
microglial cells (macrophages) in
the CNS

• Host cell activation is CRITICAL

to the pathogenesis of HIV,
because viral DNA cannot
integrate efficiently into the
host cell’s genome unless
activation has occurred

• Activation of the host cell is also

required for initiation of
transcription of the integrated
proviral DNA into either genomic
RNA or mRNA.
HIV Life Cycle
HIV & Immune System
 AIDS
Clinical Findings

• Divided into acute, latent and late phases

• Acute phase
– Develops within 3-6 weeks of contracting the
virus
– Initial infection is characterized by fatigue,
sore throat, and lymphadenopathy
– CD4 counts are usually normal, but the p24
antigen is increased
 AIDS Acute phase

– p24
– High RNA
levels
– CD4+ cell
counts - normal
 AIDS: Early Symptomatic
Disease
• Generalized lymphadenopathy
– The presence of enlarged lymph nodes (> 1 cm)
in two or more extrainguinal sites for more than
3 months without an obvious cause.
– This finding is not associated with an increased
likelihood of developing AIDS; however, a loss
in lymphadenopathy or a decrease in lymph node
size may be a prognostic markers of disease
progression
 AIDS: Early Symptomatic
Disease
• Other non-specific findings:
– Fever
– Weight loss
– Diarrhea
– Malaise
– A distinct group of patients with early
AIDS develop immune thrombocytopenic
purpura (ITP) due to the presence of
anti-platelet antibodies.
 AIDS
Laboratory Findings
• Antibodies against gp120
and gp160 (ELISA test)
are not usually present
for approximately 4-8
weeks (”window period”)
– screening tests in
blood banks may be
negative during this
phase
• Positive ELISA screens
are confirmed by the
Western blot analysis
which detects p24 and
gp41 antibodies in order
to increase overall
specificity Testul Western Blot. Line 1: CTR pozitiv, 2: CTR negativ, 3-4: teste
pozitive HIV-1, 5-6: teste pozitive HIV-2, 7-10: teste pozitive seriate la
un pacient infectat HIV-1
 AIDS
Asymptomatic latent phase
– Virus is actively
proliferating and is
present in follicular
dendritic cells
(macrophages) in
lymph nodes
– The rate of disease
progression
correlates with HIV
RNA levels
– The CD4+ cell counts
fall progressively
during this
asymptomatic period
at the rate of
approximately 50
cells/microliter/year
 AIDS
Symptomatic phase
• Clinical Findings
– Follows an average span of 10 years
– CD4 count drops to below 400 cells/uL
– p24 antigen appears again
– Patients develop minor opportunistic infections,
but are not severe enough to be indicative of a
defective cell-mediated immune response
– Oral lesions: Thrush, hairy leukoplakia and
aphthous ulcers are very common during this
stage. The first two are associated with declining
immunologic function (< 300 CD4+ T cells)
 AIDS
Advanced AIDS
• Diagnosis of AIDS is based upon an opportunistic
infection (usually Pneumocystis carinii pneumonia)
and/or a CD4 count of  200 cells/uL in an HIV
positive individual
• Most common cause of death today is bacterial
pneumonia (most common pathogen is Mycobacterium
avium intracellular –MAIC- causing bacterial
pneumonias), disseminated CMV infections, and
Streptococcus pneumoniae
• Due to the use of trimethoprim and aerosolized
pentamidine, Pneumocystis carinii is no longer the most
common cause of death
• Average life span from beginning of infections to death
is 10 years
 AIDS
Laboratory Abnormalities

• To detect Antibodies against gp120 and

gp160
– Detected by EIA (enzyme-linked immunoassays)
within 4-12 weeks after contracting the virus)
– False negatives in patients who are
immunocompromised or who have serum
collected before the antibody has developed
– False positive in patients with autoimune
diseases, hepatitis, receiving recent vaccins,
with anti MHC II Ab
 AIDS
Laboratory Abnormalities
• AIDS antibodies • Other laboratory
– Duplicate negative tests
are reported as negative abnormalities
– Second test turning
positive must be – Reversal of the CD4/CD8
verified by obtaining suppressor T cell ratio
positive results in 2 out
of 3 repeated tests – Absolute lymphopenia
– All positive or
indeterminate EIA tests – Hypergammaglobulinemia
are confirmed with a secondary to polyclonal B
Western blot assay
which measures p24 and cell stimulation by EB
gp41 antibodies to virus and CMV
improve the overall
specificity of the test – Decreased mitogen
• A positive EIA in
combination with a positive blastogenesis
Western blot has a positive – Anergy to skin testing
predictive value of 99.5%
Western Blot Results
 AIDS
Laboratory Abnormalities
• Other laboratory
abnormalities
– Decreased production
of cytokines
– NK cells are not
activated since gamma
interferon from CD4
cells is decreased
– Increased p24 core
protein indicates
active disease and has
two peaks; one during
the acute infection
and the other at the
onset of AIDS