[IMS | Lec | April 1]

ANTIBODY DIVERSITY
- A human can make 1015 different types of antibodies in terms of antigen binding specificity.

STRUCTURE AND EXPRESSION OF IMMUNOGLOBIN GENES

Two Types of light chain:
1. Kappa Genes- Chromosome 2
2. Lambda Genes- Chromosome 22

GENE SEGMENTS:
Chr-2 K Light Chain (Gene-VJC)
• V-40
• J-5
• C-1
Chr-22 Lambda Light Chain (Gene-VJC)
• V-31
• J-4
• C-7

Chr-14 Heavy Chain (Gene-VDJC)

• V-51
• D-27
• J-6
• And a series of C gene segments

Light Chain
• V & J- Codes for the variable region of the light chain
• C- Codes for the constant region of the light chain
• B cell Maturation- Genes undergo recombination

• V-J Joining
a. V-40
b. J-5

• V-J-C Gene
i. C (mu, delta etc.)
ii. Mu (First to appear)
iii. Delta
• VJC= Variable region
• Undergoes transcription and then translation into a functional K light chain.
• Exons- Coding genes
 • Introns- Non-coding Genes
• Remember: Rearrangement of the kappa gene prevents the other chromosome from re-arranging;
only when there is a none functioning gene product arising from kappa rearrangement will a
lambda chain synthesis occur
• Kappa- 60%
• Lambda- 40% (Konti dahil maactivate lang ang Kappa kapag may problem sa functionality ni
Kappa)
• If a successful rearrangement of DNA on one chromosome occurs, then the genes on the second
chromosome are not rearranged; this phenomenon is known as ALLELIC EXCLUSION.

Heavy Chain
• Synthesis same as the light chain but encoded by 4 genes (VJDC).
• B Cell Maturation
a. D-JV
b. V-D-J Gene Segment C (VDJ is the variable region)
• IgD- Has no immune function.

Summary
Light chain
a. Occurs after the rearrangement in the heavy chain.
b. Similar in pattern
c. No D-genes involved.
• Remember L chain rearrangement occurs only after the Mu chain appears.
• Mu chain synthesis is a pivotal step in the L chain process. (Because of proximity to J
chain)
• Class switching: conversion of an immunoglobin from one isotype to another while
retaining the same antigen specificity.
• Switching is dependent on antigen stimulation.
• T cells and cytokines will alter the structure making the switch site accessible to
recombinase enzymes
• Class switching occurs in activated B cells (Memory Cells) and not in naïve B cells.
 • Class switching involves only the heavy chain.

Combination of Heavy & Light Chains Adds the Final Diversity of Variable Region
• 8262 possible heavy chain combinations
• 320 Light chain combinations
• Over 2 million combinations
• P and N nucleotide additions and subtractions multiply this by 10 4
• Possible combination over 1010

[IMS | Lab | April 1 - SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES]

Serology
• The scientific study of blood sera and their effects
• Subdivision of immunology concerned with in-vitro Ag-Ab reaction.
• Concerned with the laboratory study of the activities of the components of serum that contribute to
immunity.
Immunology
• The study of molecules, cells, organs, and systems
• The desirable and undesirable consequences of immune interactions.
• The ways in which the immune system can be manipulated to protect or treat disease.
• Affinity of Interaction-The combined strength of non-covalent interaction
• Avidity- multiple interactions between the multivalent antigen and antibody

Cross Reactivity
• Presence of antigenic determinants that resemble one another so closely that antibody formed
against one will react with the other.
• Most cross-reactivity can be avoided through the use of monoclonal antibody directed against an
antigenic determinant that is unique to a particular antigen.

3 Zones of Precipitation Reaction in Fluid

Zone of antibody excess: precipitation is inhibited, and the antibody does not bind to the antigen
and can be detected in the supernatant.

Zone of equivalence: ag-ab binding is optimum, and precipitation shows.

Zone of antigen excess: precipitation is inhibited, and antigens not bound to antibodies can be
detected in supernatant.

Method
• Ab in gel
• Ag in a well
1. Wells are cut in the gel.
2. Reactants are added to the well.
3. Incubate up to 48 hours in a moist chamber.

Diffusion Payters
• Fusion of lines at their junction to form and are: Serologic identity/ presence of common epitopes.
• Crossed lines.
a. Demonstrate to separate reactions.
b. Compared antigens shared no common epitopes.
• Fusion of 2 lines with spur- Partial identity

Immunoelectrophoresis
• Double diffusion techniques that utilize electric current to enhance results.
• SPEED, Specificity
• Introduced by Grabar and Williams in 1953
• Combine immunodiffusion.

Rocket Immunophoresis (Laurel Technique)

• One dimension
• Adsorption of RID
Complement of fixation
Neutralization
• The ineffectivity of an organism or the activity of a toxin

[IMS | Lec | April 8, 2024]

Cytokines
• Regulate the immune system
• Chemical Messenger
• Are induced in response to specific stimuli.
• Effects: regulation of growth, differentiation, and gene expression
• Hormone-like substance
• Soluble molecules, some are membrane-bound molecules.
• Chemically they can be protein or glycoproteins.
• Molecular mass is less than 30 kDa.
• Have a short life.
• Produced and secreted by a variety of cells.
• Act as an intracellular mediator.
• Cytokine binds to specific receptors on the surface of other cells.
• (If it binds) It Alters the activity of cells.
✓ Cell may prepare to divide.
✓ Undergo growth and differentiation.
✓ Secrete its cytokines.

Mode of Action
Structural Grouping
1. Interferons
2. Tumor Necrosis Factor
3. Interleukins
4. CSF
5. Transforming Growth Factor

Functional Groupings
• Mediators and regulators of innate immunity: IL-1, TNFα, IL6, interferonα/β, etc. Act on endothelial
cells and leukocytes and stimulate early innate response.
• Mediators and regulation of adaptive immunity: IL-2,IL-4,IL-5,TGF-β. Act on lymphocytes to
stimulate and regulate adaptive responses to specific things.
• Stimulators of hematopoiesis: Stem cell factor, IL-3, IL-7, GM-CSF. Act on the bone marrow to
stimulate the growth and differentiation of lymphocytes.

Interleukins (ILS)
• Produces by one leukocyte acting on another leukocyte.
• About thirty-five interleukin (35) s have been identified
• They are named interleukin followed by numbers.
• IL-1 to IL-35

Tumour Necrosis Factors (TNFs)

• Activate neutrophils.
• Produced by mast cells, macrophages, and T cells.
• Firmly anchored into the cell membrane.
• Regulate immune responses and inflammation.
• Responsible for apoptosis of many cell types.

Interferons (IFNS)
• Names because of their ability to interfere with viral replication.
• Two Types:
1. Type I: IFN alpha, IFN beta (Produced in response to viral infection)
2. Type II: IFN gamma (Increased phagocytosis by macrophage)

Colony Stimulating Factor

• Essential for the growth and differentiation of immature leukocytes in the bone marrow
• Ensure that the body is supplied with sufficient white blood cells of all types.
✓ SCF: Stem Cell Factor
✓ GM-CSF: Granulocyte Macrophage-CSF
✓ G-CSF: Granulocyte-CSF
✓ M-CSF: Monocyte-SCF
✓ EPO: Erythropoietin

Chemokines
• Nagsasabi kung nasaan ang pathogen
• Act as chemo…
Effects
• The effects of cytokines are often pleiotropism, redundancy, synergy, and antagonism, which
form a cytokine network.
(Read more:https://www.slideshare.net/santupolley/cytokines-33554318)
• Pleiotropism- refers to the ability of one cytokine to have multiple effects on diverse cell types.
• Redundancy- refers to the property of multiple cytokines having the same or overlapping
functional effects.
• Synergy- refers to the property of two or more cytokines having greater than additive effects.
(Increased expression of Class I MHC molecules on many cell types)
• Antagonism- refers to the ability of one cytokine inhibiting the action of another.
• Cascade Effect- cytokines can stimulate the production of other cytokines.

Biological functions of cytokines

1. Take part in non-specific immunity.
2. Take part in specific immunity.
3. Stimulate hematopoiesis.
4. Take part in inflammatory reaction.

For cytokines, please refer to 87-93 (Turgeon)

[C-REACTIVE PROTEIN]
• C1q- magrerecognize ng pathogen
• No circadian rhythm
• Not affected by food intake
• Chromosome 1

Synthesis:
1. Macrophage
2. IL-6
3. Liver
4. Pentameric C Reactive Protein
5. T cell

CRP AND CVD

• Level of C-reactive protein can be an indicator of how at risk you are for developing CVD problems
• Low Risk- because of low cholesterol levels and little history of risk factors that’s is no diabetes,
high blood pressure or smoking and no family history of heart attacks at an early age. Even higher
level of CRP will probably not put you at a level of risk that requires than lifestyle changes
• Intermediate Risk- helpful to know CRP
• High Risk- Not essential to know your CRP, because you should already be combining aggressive
treatment with lifestyle changes to lower your risk
 • High circulating CRP levels put an apparently healthy Individual at an Increased risk of congestive
heart failure
• Risk
• Low less than 1.0 mg/L
• Average 1.0-3.0 mg/L
• High greater than 3.0 mg/L
• When an individual's concentration is 5.0 mg/L, or above an almost 3 fold increased risk of heart
failure is present

RADIAL IMMUNODIFFUSION ASSAY

• Used for the quantitative determination of human CRP in serum
• Human CRP is added to agar well plates of decreasing amounts of human anti-CRP antibiodies
• Sera diffuses through the agar and forms a precipilation ring with the antibodies.
• The diameter of the ring is compared with a standard to determine the amount of CRP in the blood
• Disadvantage: Low sensitivity

High Sensitivity CRP Enzyme Immunoassay Test (hs-CRP ELISA)

• Able to detect small quantities of CRP in the serum unlike the RID assay
• Sold phase enzyme linked immunosorbent assay, sandwich method
1. Mouse antibodies against human CRP are attached to the wall of the well plate.
2. Human CRP and goat antibody against human CRP are added to the well plate.
3. A sandwich structure is formed with the two antibodies and the human CRP
4. All unattached antibodies are washed away.
5. Tetramethylbenzidine is added to the plate resulting in a blue color.
6. HCL is then added to change the blue color to yellow. This is the stopping agent.
7. The amount of CRP is determined by the intensity of the color through spectrophotometric
devices at 450nm.

Conclusion
• CRP is an acute phase reactant in response to inflammation.
 • It is suspected to play a role in diseases such as cardiovascular disease.
• The levels of CRP in the blood help indicate one's risk for disease. The higher the level, the higher
the risk for disease.
• The levels are measured by high-sensitivity tests such as the ELISA
• Researchers are working to find out more about CRP's pathogenesis.

[SEROLOGICAL DETECTION OF STREPTOCOCCAL ANTIBODIES RHEUMATOID FACTOR]

STREPTOCOCCAL SEROLOGY
• Streptococci are gram (+), beta-hemolytic, spherical, ovoid, or lancet-shaped organisms which are
catalase negative and seen in pairs or chains
• Divided into groups or serotypes based on cell wall componentsàStreptococcus pyogenes belongs
to Lancefield group A and it is believed the M protein is the chief virulent factor of this group
• Numerous exo-antigens are produced and excreted as the cell metabolizes (Streptolysin 0, DNase,
Hyaluronidase, Nicotinamide, Adenine
dinucleotidase (NADase), Streptokinase)
Culture and rapid screening tests detect early Infection
• Sequelae include Rheumatic Fever and Acute GN

Structure of Streptococci

GROUP A STREPTOCOCCAL INFECTION

• Two major sites of Infection: upper respiratory tract and skin
 • Upper respiratory tract = sore throat, tonsillar exudate
• Skin pyoderma or impetigo
• Suppurative complications = erysipelas,scarlet fever, septic arthritis, meningitis •
• Non-suppurative complications = RF or Post- streptococcal GN

A. Rheumatic Fever
• only certain serotypes of S. pyogenes is involved
• develops as sequelae in 2-3% untreated upper respiratory Infections
• symptoms occur about 18 days after sore throat
• Group A streptococcus share antigenic determinants with host tissue, especially
• heart and even joints inflammation of mitral valve most serious
• 30-60% of patients may suffer permanent disability

B. Post-Streptococcal Glomerulonephritis
• follows Streptococcal infection of skin or pharynx
• occurs about 10 days following Initial infection
• characterized by damage to glomerull of the kidneys
• renal function Impaired due to reduction In glomerular filtration rate, results in edema and HPN
• renal failure not typical
• one theory is damage caused by antigen- antibody complexes depositing in kidneys

LABORATORY TESTING
• Most reliable test is culture and identification of the organism from infected site
• Rapid streptococcal screening tests from the throat exudates have high specificity but low
sensitivity, 60-85%
• Detection of Streptococcal antibodies most useful in Streptococcal sequelae• The most useful
antibodies are: ASO, anti-DNase B, anti-NADase, anti- Hyaluronidase
• Serological evidence of disease is based on elevated or rising titer of Streptococcal antibodies
• Four-fold (2 tube dilution) rise in titer is considered clinically significant
Anti-Streptolysin O Titer (ASO Titer)
 • Two of the toxins produced are Streptolysin S, which is oxygen stable, non-antigenic and
Streptolysin O (SLO), which is oxygen labile and antigenic
• SLO is a hemolysin which is toxic to many tissues, including heart and kidneys
• Evokes an antibody response (anti-SLO) which neutralizes the hemolytic action of SLO
• The test is specific for ASO, It does not test for antibodies to any other Streptococcal exotoxins
• Normal values will vary, <125 Todd units for adults, 5-125 Todd units for children, recent Strep
Infections 250 Todd units for adults, 333 Todd units for children
• A single titer is of little significance unless extremely elevated, titers performed over a period of
time will give the most information
• ASO TITER increase within 1 to 2 weeks after infection
• Peak between 3 to 6 weeks following the initial symptoms (sore throat)

1. Nephelometric method
• antigen used, purified recombinant streptolysin
• when antibody positive patient serum combines with the antigen reagent, immune complexes are
formed resulting in an increased light scatter that the instrument converts to a peak rate signal
2. Anti-DNase B Testing
• may appear earlier than ASO
• Increased sensitivity for detection of glomerulonephritis preceded by streptococcal skin infection
• macro- and micro-titer, ELISA, and neutralization available techniques are
• Neutralization technique has advantage of stability of reagents
• DNase B is an enzyme produced by almost all strains of beta hemolytic Group A and a few strains
of Group C and G streptococci.
• Patients infected with these strains of streptococci form antibodies against DNase B. Serologic
tests for anti-DNase B are helpful in confirming the diagnosis of streptococcal infections.
• anti-DNase B test is an enzyme neutralization test, measuring the ability of specific antibodies to
inhibit the depolymerization of DNA
• The reciprocal of the highest dilution of serum showing inhibition is the anti-DNase B titer.
• Elevated titers are found in approximately 80% of acuto theumatic fever cases.
• An elevated anti-DNase B titer is helpful in diagnosing acute rheumatic fever in the 20% of patients
who do not have elevated ASO titers.
• Anti-DNase B titers rise more slowly than ASO titers, peaking four to eight weeks after infection.
They also decline much more slowly, remaining elevated for several months.
• Following streptococcal pyoderma, which may result in post streptococcal glomerulonephritis, less
than 50% of patients develop elevated ASO titers. In contrast, most patients develop elevated anti-
DNase B titers, which remain elevated for many months.
• Therefore, anti-DNase B is the preferred serological test.
3. Anti-Hyaluronidase Testing

• test patient serum for antibodies which inhibit action of Hyaluronidase

 • after performance of the test, a clot will form into the tubes where enzyme activity of Hyaluronidase
has been neutralized by patient antibody
• Hyaluronidase produced by patients with throat or skin infections, ASO produced In response to
throat infections only
4. Streptozyme Testing
• hemagglutination procedure to detect antibodies to numerous Streptococcal
• antigens
• sheep RBC's are coated with Streptolysin, Streptokinase, Hyaluronidase, DNase, and NADase
• patient serum diluted 1: 100, mixed with sheep RBC's and observed for agglutination
• rapid and simple to perform, more false positive and negative results occur

RHEUMATOID FACTOR (RF)

• This test is done to diagnosed Rheumatoid arthritis, which is one of important autoimmune
disease.
• RF is an antibody (IgM or IgG classes) bind to the Fc portion of other IgG molecules, and form IgG-
anti-IgG complexes in the circulation or joint fluid.

• RFs are detected in serum in up to 80% of adult patients with RA.

• RFs are not specific for RA and occur in other autoimmune disease, in chronic infectious diseases,
such as infective endocarditis, tuberculosis, and hepatitis B.
• usually at low titer, in up to 20% of overtly normal elderly individuals

General Feature of Rheumatoid arthritis

• RA is a systemic chronic inflammatory disease of unknown etiology.
• is characterized by polyarthritis which may be progressive and permanently deforming and by
extra-articular manifestations(rheumatoid nodules, pericarditis, and arteritis).
• Adult RA is commonly associated with rheumatoid factors.
• Symptoms of Rheumatoid Arthritis
• Symptoms first begin in the small joints of the fingers, wrists and feet, with warm, swollen and
tender joints that are painful and difficult to move.
• Joints of both sides of the body (symmetrical) are typically affected.
• People with RA often experience fatigue, loss of appetite and low-grade fever
 • There is often stiffness in the morning that lasts for several hours or more.
• Nodules may form under the skin, often over the bony areas exposed to pressure (such as the
elbows).
• Over time, damage to the cartilage and bone of the joints may lead to joint deformities.

✓ Rheumatoid arthritis (late stage)

✓ Boutonniere deformity of thumb

✓ Ulnar deviation of metacarpophalangeal joints
✓ Swan-neck deformity. of fingers

Diagnosis of rheumatoid arthritis

• medical history and physical examination, looking for distribution of joints affected, joint swelling,
warmth and range of motion, motion, as well as the presence of nodules under the skin.
• Imaging studies such as X-rays, sonograms or magnetic resonance imaging may be used to detect
the degree of joint involvement or joint damage.
• A blood test can indicate the presence of an rheumatoid factor, which is found in 80 percent of
people with RA.

Methods used to detect RF

• Latex agglutination method- mixes the blood being tested with (latex) beads that are covered
with human antibodies. If rheumatoid factor (RF) is present, the latex beads clump (agglutinate).
• Haemagglutination test-mixes the blood being tested with a sheep's red blood cells that have
been covered with rabbit antibodies. If RF is present, the red blood cells agglutinate.
• Nephelometry test-Using an automated machine based on laser light scattered.

Interpretation of the test

 • Agglutination test is positive. Do titration and determine the end of titration.
• Normal range differ from lab to another, but in most lab titration of >1:20 consider positive.
• Positive test in 80% of Rheumatoid Arthritis.
• It also positive in other autoimmune disease.
• Positive in viral hepatitis.
• Positive in TB.

Factors interfere with positive result

• Hyperlipedemia- Blood that is very high in fats.
• Age- About 5% to 10% of people over age 65 have an elevated RF level.

Next Meeting: Quiz about Lab ;Lecture Only (April 15-26)

1. Syphilis
2. PTHA
3. VDRL
4. Strip
5. RF
6. CRP
7. ASO

SYPHILIS
• Previously called “great pox” or “evil pox”
• Brought from New world to Old world (NOSy)
• Old world to New world: small pox
• French, Italian, Spanish disease
• Causative agent: T. pallidum subs. pallidum

Mode of transmission:
1. Sexual
2. Parenteral (infected blood/needles)
• destroyed by refrigeration for 3 days
3. Mother to fetus (congenital syphilis)
Hutchinsonian triad
• notched teeth
• keratitis
• deafness

Congenital Infection (TORCHS)

TO-xoplasmosis
R-ubella
C-MV
H-erpes
S-yphilis

Note:
• duodenale - nematode parasite associated w/ congenital infection
• CMV - most common cause of congenital infection
Diagnostic test
• Wasserman tests (1906) - used cardiolipin antigen
• Salvarsan 606 (arsphenamine)

Treatment
1. Heavy metals
• arsenic (garlic on breath, metallic taste, affinity to keratin)
• salvarsan 606 (arsphenamine)

2. Drug of choice
• penicillin G

Stages of Syphilis
1. Primary syphilis
Lesion: hard chancre (painless)
Lab: darkfield microscopy

2. Secondary syphilis
• condylomata lata, generalized rash (wart-like lesion)
• systemic dissemination of organism

Lab:
a. darkfield microscopy
b. serological test

3. Latent syphilis
• absence of clinical signs
• Lab: (+) serological tests

4. Tertiary syphilis
• Granulomatous lesion: gummas, neurosyphilis, cardiovascular disease

• Gummas - localized areas of granulomatous inflammation

• Cardiovascular complications - aortic aneurysm, aortic regurgitation,
• angina pectoris
• Neurosyphilis - can span all stages of disease

Parenchymatous Neurosyphilis
• Paresis - incomplete paralysis
• Tabes dorsalis - degeneration of the dorsal columns of the spinal cord and sensory nerve trunks
 • Specimen: CSF
• Lab: Serological test

Laboratory Diagnosis
1. Direct observation of organism
a. Darkfield microscopy (Primary, Secondary)
• Result: coiled organism w/ corkscrew motility
• False negative results:
• Delay in evaluating the slides
• Insufficient specimen
• Pretreatment with antibiotics
b. Fluorescent Antibody testing
• sensitive an highly specific
• live specimens are not required
2. Serologic test
• STS: Serologic Test for Syphilis
• 1st: Wasserman test
• Principle: complement fixation
• Positive: no hemolysis
• Negative: hemolysis

NON-TREPONEMAL TEST (anticardiolipin/antilipoidal ab)

• Screening tool for syphilis
• Monitoring disease progress and treatment outcomes
• Used for diagnosing congenital syphilis and neurosyphilis
• nonspecific; soluble antigen
• detect the presence of reagin - antibody to cardiolipin
• subject to biologic false positive eg. SLE, RA, IM, pregnancy

Cardiolipin
• lipid material released from damaged cells; reagin reacts with lipid antigens from animal tissue
(beef heart) in a process knows as flocculation

Flocculation
• Precipitation that occurs over a narrow range of antigen concentrations; antigen consist of very fine
particles that clump together in a positive reaction

I. VDRL (Venereal Disease Research Laboratory)

• Qualitative/quantitative slide flocculation test
• Principle: Flocculation
• Specimen: 50 uL or 0.5 mL serum/CSF
• Heat: 56c for 30 mins - inactivate complement
• If >4 hours have elapsed after inactivation, reinactivate by heating to 56c for 10 minutes
• Reagent: VDRL antigen
 • Antibody: Anti-cardiolipin
• Antigen: 0.03% cardiolipin, 0.9% cholesterol, 0.21% lecithin

a. Cardiolipin - main reacting component

b. Cholesterol - enhances reacting components of cardiolipin
c. Lecithin - removes anti-complimentary activity of cardiolipin

VDRL NEEDLES
1. Qualitative Serum VDRL test
• Ring: 14 mm diameter
• Antigen Delivery: 18 gauge (60 drops reagent/mL)
• Rotation: 180 rpm for 4 mins
• Microscopic: flocculation
• Reactive: presence of medium to large clumps
• Weakly Reactive: small clumps
• Nonreactive: no clumps

Note:
• If Qualitative Serum VDRL test is positive or weakly positive perform
• Quantitative Serum VDRL test

2. Quantitative Serum VDRL test

• Ring: 14 mm diameter
• Antigen Delivery: 19 gauge (75 drops reagent/mL); 1/75 mL
• Saline Delivery: 23 gauge (100 saline/mL); 1/100 mL
• Rotation: 180 rpm for 4 mins

3. Quantitative CSF VDRL test

• Rings: 16 mm diameter
• Concavity: 1.7 mm deep
• Antigen Delivery: 21 or 22 gauge (100 drops/mL); 1/100 mL
• Rotation: 180 rpm for 8 mins

Note:
• Total magnification: LPO (100x)
• Reactive: medium to large clumps
• Weakly Reactive: small clumps
• Nonreactive: no clumps
• All sera with tractive or weakly reactive results must be tested using the quantitative slide test
• Twofold dilutions of serum ranging from 1:2 to 1:32 are initially used

II. RPR (Rapid Plasma Reagin)

• Principle: Charcoal agglutination
• Specimen: 50 uL or 0.05 mL serum
 • Reagent: modified VDRL antigen

• Antibody: Anti-cardiolipin
• Antigen: Cardiolipin

a. Cardiolipin - main reacting component

b. Cholesterol - enhances reacting components of cardiolipin
c. Lecithin - removes anti-complimentary activity of cardiolipin
d. Charcoal - easier visualization
e. Choline Chloride - for chemical inactivation of serum
f. EDTA - prevent oxidation of lipid
g. Thimerosal - preservative

• Ring: 18 mm diameter
• Antigen delivery: 20 gauge (60 drops reagent/mL); 1/60 mL
• Rotation: 100 rpm for 8 mins

• Macroscopic: clumping
• Reactive: medium to large clumps
• Weakly reactive: small clumps
• Nonreactive: no clumps

Note:
• Prozone - antibody test (false negative)
• negative but w/ rough or granular appearance
• Remedy - serum dilution
• III. USR (Unheated Serum Reagin)
• IV. RST (Reagin Screen Test)
• V. TRUST (Toluidine Red Unheated Serum Test)

• NEXT: confirm w/ treponemal test

TREPONEMAL TEST
• Confirmatory test for syphilis
• Establish diagnosis in late latent syphilis
• detect for treponemal antibodies
• specific

1. TPI (Treponema Pallidum Immobilization Test)

• standard test to w/c other treponemal tests is evaluated in live
• organism testicular chancre of rabbit
• Specimen: patent serum
• Positive: >50 organism immobilized

Note
a. Positive: >50%
b. Doubtful: 20-50%
c. Negative: <20%

II. FTA-ABS (Fluorescent Treponemal Antibody Absorption Test)

• detects treponemal antibodies in patient serum
• Specimen: patient serum
• Heat: 56c at 30 mins (inactivate complement)
• Sorbent: Reiter’s - removes cross-reactivity w/ other treponemes
• *Indirect Immunofluorescence
• Antigen: Nichol’s strain
• Antibody: anti-treponemal
• Labeled reagent: AHG FITC

• Negative: no fluorescence
• Reactive: 2+ or above
• If result is 1+, repeat with a second specimen drawn in 1 to 2 weeks

III. (HATTS) Hemagglutination test

a. HATS (Hemagglutination Treponemal test for Syphilis)
• Uses glutaraldehyde-stabilized turkey RBCs

b. TPHA (Treponema Pallidum Hemagglutination)

• Uses tanned sheep RBCs coated with antigen from Nichols strain

c. MHA-TP (Microhemagglutination)
• Most widely used hemagglutination test for syphilis
• Replace by TPPA test; uses microtiter plates
• Antigen: Sheep RBC sensitize w/ Nichol’s strain
• Antibody: Anti treponemal

d. TPPA (Treponema Pallidum Particle Agglutination)

• Antigen: Gelatin particles sensitized w/ Nichol’s strain of treponema
• pallidum
• Antibody: Anti treponemal

Situation:
1. (-) RPR, (-) FTA-ABS: no syphilis
2. (+) RPR, (-) FTA-ABS: biologic false positive ex. SLE, IM,
Pregnancy
3. (-) RPR, (+) FTA-ABS: late/treated Syphilis

Note:
Non-treponemal test (VDRL & RPR)
• test to assess reinfection

1. Traditional Algorithm: non-treponemal test to treponemal test

 • recommended by CDC

2. Reverse Algorithm: treponemal test to non-treponemal test

• detect early/late/treated syphilis
• higher rate of false positive

OTHER DISEASES
• Yaws: T. pertenue
• Pinta: T. carateum
• Bejel or endemic syphilis: T. pallidum endemicum
• Rabbit syphilis: T. cuniculi (Wasserman NR)

Quick Notes:

TPHA
• QUALITATIVE
• DETECT IgG + IgM ANTIBODIES TO syphilis IN serum OR EDTA plasma titer LEVEL

SYPHILIS
• SPIROCHAETE TREPONEMA PALLIDUM
• MOT: SEXUAL/INFECTED BLOOD
• CHANCRES - INITIAL FORES
• Syphilitic rash à CVD

NEWBIO TPHA
• AVIAN ERYTHROCYTES COATED W/ ANTIGENS OF T. PALLIDUM
• NICHOLS STRAIN
• TEST CELLS: Avian erythrocyte COATED W/ PALLIDUM AG
• CONTROL CELLS: Avian ERYTHROCYTE
• SAMPLE DILUENT: Saline SOLUTION W/ ABSORBENTS
• (+) CONTROL: Human antiserum titre 1/1280
• (-) CONTROL: Rabbit SERUM
• 96 well - U well microplates

STORAGE
• Test & Control cells store upright @ 2-8°C
• Test, Control, Sample Diluent and Controls are stable up to 3 months when stored properly

PRECAUTION
• IN VITRO ONLY
• Test, Control, Sample Diluent and Controls has sodium azide as preservative
• Control - human / animal origin
• REAGENT - ANIMAL ORIGIN
• Resuspend Test & Control before use
 • IF NOT, INADEQUATE DILUTION & erroneous RESULTS
• Cover controls with medium before storage
• IF NOT, CLUMPING IN TEST WELL

SAMPLE COLLECTION
• Human serum/EDTA plasma
• UP TO 7 DAYS AFTER COLLECTION
• Visible components are removed by centrifugation
• Store 2-8°C up to 7 days
• Can be frozen at < -20°c for up to 1 month
• THAWED UP TO 5 TIMES

PROCEDURE
• Each sample - 3 wells
• DILUTION 1:20
• 190 ul diluent: 10 uL sample

TEST
• 25 UL +)+) CONTROL
• 25 NL DILUTED VX TEST WELL
• - CONTROL HELL
• 75 NL TEST CELL TO (6) + (-) CTRL WELLS

FOR DILUTED SX
• 75 uL Test Cell to Test Well
• 75 uL Control cell to Control well
• Final sample/Control Dilution: 1:80
• Incubate 15-30°C; 45-60 mins

SAMPLE TITRATION AUSAY

• Each sample: 9 wells
• 1 in 80 – 1 in 10240
• Positive and Negative Control: 2 wells
• Control Cells: 1 well

1. Dilution (same above)

2. Titration
• Empty 2nd and 3rd wells
• 25 uL diluent well – 4-10
• 25 uL sample dilution to 2nd and 3rd wells
• 25 uL sample dilution to 4th well
• Mix and Serial Dilute

3. Test
• Resuspend Test and Control
 • 75 uL Control Cells – 2nd well
• 75 uL Test Cells – 3rd to 10th well
• Mix
• Incubate 15-30 C for 45-60 minutes
• Read agglutination pattern
• Sample Titer: Reciprocal of Final Positive Sample Dilution

4. Control Procedure
• Kit (+) End Point: 1/640-1/2560
• TPHA Control: Pre-diluted
o Added directly to well w/o diluted
• TEST CELLS ADDED DIRECTLY TO CONTROLS

INTERPRETATION
• Negative: Non reactive
o Reactivity < Equivocal
• Positive: Reactive/Equivocal
o Repeat in Duplicate
o Either (+): (+)
o Both Non Reactive: (-)
• Reactive Sample in Test and Control Cells
o Agglutination is greater than in Test Cells: (+) and Repeat

Sample Titration
• Titer > 1/80 - Reactive
o Repeat in Duplicate
o Reactive: Active, Past, Successfully treated syphilis

Absorption of Non-Specific Reactions

• Sample greater/equal agglutination in Control than Test Cell

Steps
1. 10 uL sample to 190 uL control cells
2. Centrifuge: Min. 1500g for 3 minutes
3. 25 uL supernatant from step 2 to each 2 wells
4. Ensure test and control are resuspended
a. 75 uL Test Cell – Well 1
b. 75 uL Control Cell – Well 2
5. Mix and Incubate 15-30 C for 45-60 minutes
6. Interpret

REPRODUCIBILITY
• Mixed titer panel
o 25 – Positive Syphilis sample
o 5 – Negative Syphilis sample
• 5 testing days over 7 days in duplicate
 • 2 separate runs each day

Prozone
• High antigen levels

Syphilis Agreement
• Positive: Agreement = 250; 98.54-100%
• Negative: Agreement = 50; 92.89-100%
• Overall: Agreement = 300; 98.78-100%

BLUE SCREEN SYPHILIS ULTRA RAPID TEST STRIP

• Detect IgG and IgM antibody
• Qualitative
• Membrane strip
• Whole Blood, Serum, Plasma
• Chromatogenic

Syphilis
• Long latent
• Asymptomatic
• Primary Syphilis: Chancre
• Antibody Response: Detect within 4-7 days after chancre

Principle
• With Treponemal antibody: Red Line in Test Line (+)
• Control: Pink line in Control Line

Reagent
• Test Strip
o Syphilis antigen coated particle
o Syphilis antigen coated on membrane

Precaution
• In vitro
• Affect results: Humidity and Temperature

Storage
• Room/Refrigerated Temperature (2-30 C)
• Test Strip – sealed pouch; Do not freeze

Sample
• Non hemolyzed
• Serum or Plasma
o 2-8 C up to 3 days
o -20 C long term
 • Whole Blood
o 2-8 C; run within 2 days
o Do not freeze

Use
• Best results: Within 1 hour
• Card: Strip in the middle; arrows downward

Serum or Plasma
• 2 drops (approx. 50 uL)
• 1 drop buffer (approx. 40 uL)

Whole blood
• 2 drops
• 1 drop buffer

Fingerstick/Capillette

1. Capillary Tube
• Approx. 50 uL whole blood
• 1 drop buffer

2. Hanging Drop
• 2 hanging drop (approx 50 uL)
• 1 drop buffer
• Read at 10 minutes; Do not read after 30 minutes

Result
• Positive: 2 red lines (control and test line)
• Negative: 1 red line (control line)

Red Intensity: depends on T. pallidum antibody concentration

Invalid
• No control line
• Insufficient specimen
• Incorrect technique

Limitations
• In vitro
• Detect Treponemal antibody (Qualitative)
• Not sole criteria for diagnosis
• Accuracy > 99.7%
• Sensitivity: 99.7%
• Specificity: 99.6%
ANTISTREPTOLYSIS O (ASO) REAGENT SET LATEX SLIDE TEST

Use
• Qualitative and semi-quantitative measurement of antibodies to Streptococcal exoenzymes in
human serum

Todd (1932)
• Discovered Streptolysin O

Principle
• Based on reaction between Streptococcal exoenzymes bound to latex particles and Streptococcal
antibodies in the sample

Reagents
• Agglutination: ASO titer of 200 IU/mL or higher in serum

1. Latex Reagent
• Polystyrene particles coated with Streptococcal exoenzymes

2. Positive Control
• Human serum with at least 200 IU/mL of ASO reactive with test reagent

3. Negative Control
• Human serum with less than 200 IU.mL of ASO non reactive with test reagent

4. Glycine Buffer
• pH 8.2 + 0.1
• Diluent
o 0.1 M Glycine + 0.15 M NaCl
o All reagents contain 0.1% sodium azide (preservative)
o Store 2-8 C

Precautions
• In-vitro use
• Controls: Negative for HbsAg and HIV

Specimen
• Fresh serum; never plasma
• Serum: stored 2-8 C for less than 48 hours
• Frozen: longer period

Qualitative Test
• 1 drop positive control
• 1 drop negative control
• 1 drop serum sample
 • 1 drop latex reagent
• Rock for 2 mins

Results
• Positive: Agglutination; ASO conc: > 200 IU/mL
• Negative: No agglutination; milky suspension

Semi-quantitative Test
• At least 5 tubes
• 1 drop each controls onto slide
• 1 drop each dilution
• 1 drop latex reagent
• Rock for 2 mins

Results
• Positive: agglutination
• Titer: reciprocal of highest dilution with positive reaction
• Read at 2 mins

IU/mL sample = (+) Control Conc x Reciprocal

Upper Limit of ASO Titer Values

• Preschool Children: less than 100 IU/mL
• School Age Children: 166-250 IU/mL
• Average Adult: Less than 200 IU/mL

Above Upper Limit: Streptococcal Infection

2 Dilution rise between acute and convalescent stage: significant

ASO Titer Rise: After 1 week

Maximum level: 3-5 weeks
Return to Pre-Infection: 6-12 months

C REACTIVE PROTEIN (CRP) LATEX TEST

CRP
• Human serum acute phase
• Synthesized by hepatocytes
• Present in trace amount
• MacLeod and Avery: CRP antibody more sensitive than C-polysaccharide assay
• Assays that Measure CRP: Capillary Precipitation, Double Immunodiffusion, Radical
Immunodiffusion

Principle
• Reaction between CRP antisera bound to latex particle and CRP in test sample
 • Visible Agglutination: >0.8 mg/dL CRP mixed with latex reagent

Reagents
1. Latex reagent
• Polystyrene particles with monospecific antihuman CRP in glycine buffer
• pH 8.8 + 0.5
• Sensitivity: approx 0.8 mg/dL

2. Positive Control
• Serum with >0.8 mg/dL CRP

3. Negative Control
• Serum Non Reactive with reagent

4. Glycine-Saline Buffer
• pH 8.2 + 0.1
• 0.1 M glycine; 0.15 M NaCl
• 0.1% sodium azide preservative

Controls: negative for HbsAg

Reagent
• Store refrigerated (2-8 C)
• Refrigerated – sedimentation

Sample Collection
• Fresh serum
• Store 2-8 C for less than 72 hours
• Frozen: longer period
• Do not use plasma

Qualitative
• 1 drop positive or negative control or undiluted sample
• 1 drop latex reagent
• Rotate slide for 3 minutes
• Accuracy: 100%

Semi-Quantitative
• At least 5 tubes
• 1 drop of each control
• 1 drop of each dilution
• 1 drop reagent
• Rotate for 3 minutes
• Serum Titer (mg/dL) - (reciprocal of highest dilution with positive reaction)(positive control
concentration)
 • Precision: 92.9%

Results
• Positive – agglutination
• Negative – no agglutination; milky suspension

Values
• Healthy CRP: 0.02-1.35 mg/dL
• Mean Value: 0.047 mg/dL

Limitations
• Critical reaction time
o More than 3 minutes: false positive (drying)
• Freezing reagent
o Spontaneous agglutination
• Prozone
o False negative
o Check (-) sera at 1:10 dilution with glycine buffer
• Agglutination not indicative of CRP concentration

Sensitivity: >0.8 mg/dL

Linearity: 25 mg/dL

RHEUMATOID FACTOR (RF) REAGENT LATEX SLIDE TEST

• Ab against Ag sites at Fc of human/animal IgG
• Ability of rheumatoid arthritis sera to agglutinate sensitized sheep red cells (Waaler & Rose)
• Latex beads coated w/ human gamma globulin (Singer and Plotz)

Advantages
✓ Rapid (3 mins)
✓ Lack of heterophile Ab interference

Principle
✓ Rf reagent
✓ Polystyrene latex sensitized w/ human IgG

Sensitivity
✓ min. 8 10/ml rf
Reagent
1. Latex rgt
-Ph 8.2
2. (+) ctrl serum
-Stable prediluted human serum at least 8 IU/mL RF
3. (-) ctrl serum
-Less than 8 IU/mL RF
4. (GS) Glycine - Saline buffer
 - ph 8.2 ± 0.1 M Glycine & 0.15 M NaCl

Dilute
✓ 1 buffer 1: 19 d. H20
✓ All rgt: 0.1% sodium azide
✓ Human sera in controls
✓ Negative HbsAg and HIV
✓ STORE 2-82; NO FREEZE!

SX COLLECTION
✓ Fresh serum
✓ 2-8℃ ;<72 hrs
✓ Frozen: Longer period
✓ No to plasma!

QUALITATIVE TEST
✓ Room temp
✓ 1 drop (+) ctrl
✓ 1 drop (-) ctrl
✓ 1 drop rgt
✓ Rotate 80-100 rpm 2 mins
✓ (+) AGGLUTINATION
RF conc. ≥ 8 IU/ml
✓ (+) RUN AGAIN W/ QUANTI

QUANTITATIVE TEST
✓ Use GS buffer to dilute specimen
✓ 1 drop (+) + (-) ctrl
✓ 1 drop each dilution
✓ 1 drop rgt (sero card)
✓ 1 drop rgt (test card)
✓ Rotate 2 mins
✓ (+) AGGLUTINATION
✓ (-): NO AGG. MILKY SUPENSION

SEMI-QUANTITATIVE TEST
✓ TITER: Reciprocal Of Highest Dilution (+)
✓ 10/mL SX = IU/mL CTRL x SX TITER

LIMITATIONS
1. Read at 2 minutes
2. Prozone at high titer
Not encountered
3. RF
✓ RA
✓ Infectious mononucleos
✓ Sarcodosis
 ✓ Lupus erythematosus
✓ Sjogren’s Syndrome
4. Some RA patients have no RF in serum
SENSITIVITY: 8 IU/mL or above

[IMS | Lec | April 17, 2024]

COMPLEMENT PATHWAY
• Complement can be activated in 3 different ways
• Classical Pathway:
✓ 9 proteins involved
✓ Triggered by antigen-antibody complex
✓ Last activated
• Alternative Pathway
• Lectin Pathway
✓ Ab independent means of activating complement proteins
✓ Major constituent: mannose-binding lectin
✓ Activation of complement seldom involves only 1 pathway
✓ MBL adhere to mannose found mainly in outer cell walls or outer coating of bacteria,
viruses, yeast, protozoa

Complement system
• Plays a major part in inflammatory responses directed against foreign antigens
• End product of activation lysis of invading cell
• Complement fragments act as opsonins
• Increase vascular permeability, recruit monocytes and neutrophils to the area of antigen
concentration and trigger secretion of immunoregulatory molecules that amplify the immune
response

CLASSICAL PATHWAY
• Activate Ab-Ag
• Classical pathway is the main antibody-directed mechanism for triggering complement activation
• Not all immunoglobulin can activate this pathway
• IgM is most efficient due to its multiple binding sites
• IgM, IgG1, IgG2, IgG3

3 main stages of activation of classical

• C1 recognition unit
o Recognizes the antigen-antibody complex because it will activate classical.
• C4, C2, and C3 collectively known as the activation unit
• C5 through C9 comprise the membrane attack complex (MAC)
• When the MAC is formed, cell lysis happens
• C1 first complement component to bind
• 3 subunits: C1q,C1r,C1s
• Part of a specific immune response because it relies on antibodies to initiate it
 • C1 becomes activated when it binds to the ends of antibodies
• Antibody formation is first done before the classical pathway is activated
• Stabilized by calcium
• Generate enzyme activity to begin the cascade
• C1q, C1r, C1s – end of recognition unit
• Cleaves C4 and C2 into C4a and C2b
• Start of activation unit C4b2a

C1q recognizes the Fc region of 2 adjacent antibody molecule

Binding occurs are the CH2 region for IgG
CH3 region for IgM
C1r and C1s both serine protease proenzyme (not yet an enzyme)
Binding of C1q both C1s and C1r converted into active enzymes
Once C1s is activated the recognition stage ends

C1 complex - binds to the Fc region of the antigen-antibody complex and C1 complex

The one that binds to antibody – C1q
When C1q binds to C1r and C1s, it will generate enzyme activities to initiate the cascade.
Then that’s when C1r and C1s possess enzymatic activity
Once the cascade is formed, it will proceed to the activation stage
Antigen: pathogen; not necessarily bacteria

IgG – CH2 binding site

IgM – CH3 binding site

ANAPHYLATOXIN KININ LIKE ACTIVITY (C2B)

• C4a: anaphylatoxic activity
• Anaphylatoxins: cause smooth muscle contraction, vasodilation, histamine release from mast cells
and enhanced vascular permeability
• They also mediate chemotaxis, inflammation and generation of cytotoxic oxygen radical
• Once the pathogen is recognized by C1, it binds to the Fc region of the Ab-Ag complex
• C1r and C1s are activated and have enzymatic activity and initiate the activation unit until
recognition is finished

Activation
• C4 becomes C4a and C4b
• C4b – activates C2 and cleaves to C2a and C2b
• C4b and C2a binds
• C2b and C4a gains metabolic actions
• C3 convertase is formed (of the classical pathway)

Next phase of activation unit (STARTS SA ACTIVATION NG C4)

• Formation of activation unit: production of enzyme (C5 convertase)
• C4 – 2nd most abundant complement protein
• C4 is cleaved to C4a and C4b
 • C4b must bind to protein or carbohydrates within seconds and react with Ic4b (water) to
degrade

C2 – next component to be activated

• When combined with C4b in the presence of Mg, C2 is cleaved by C1s to form C2a and C2b
• Combination of C4b and C2a known as C3 convertase (C4b2a) indicate active enzyme
• C4b2a - not very stable complex; Half life:15 secs to 3 mins
• Should bind to C3 quickly

C3 – major constinuent of the complemnent system

• Serves as key point for all 3 pathway
• Cleavage of C3 to C3b represents the most significant step in the entire process of
complement activation
• C3b serves as a powerful opsonin
• C3b bind to C4bC2a (c5 covertase)
• Last step in the formation of the activation unit
• C3a is released which has anaphylactic activity
• C3b binds to complex = C4b2a3b (c5 convertase)

Membrane Attack Complex

• C5 convertase cleaves C5 into C5a and C5b forming the beginning of MAC
• Causes cytolysis
• The circular membrane attack complex acts as a channel in which cytoplasm can rush out and
water rushes in.

Function of C5a
• C5a disperses away from the bacteria
• Bind to mast cells and increase inflammation
• Powerful chemotactic factor known as leukocytes

Building the membrane attack complex

• C5b on the surface of bacteria binds to C6
• The binding of C6 to C5b activates C6 so that it can bind to C7
• C7 binds to C8 which in turn binds to many C9’s
• Together these proteins form a circular complex called the Membrane attack complex (MAC)
Cell membrane of bacteria – C7,C8,C9 then a hole in the cell
• Loss of electrolytes
• Those who shouldn't enter, enter
 • Loss of fluid
• The contents of the cell leak out through the MAC pore and the cell dies

ALTERNATIVE PATHWAY
• Formation of ag-ab complex (recognize cell wall of the pathogen)
• 4 components: C3, factor B, factor D and properdin
• Triggering substances may be pathogens or Nonpathogens bacterial cell wall components, fungi,
viruses, parasites immune complexes, RBCs, polymers
• C3 key component
• C1,2,4 – not used in this pathway
• C3 cleaves to C3a and C3b
• Steps:
✓ C1q will recognize the Fc region
✓ Once recognized, C1q will activate C1r and C1s
✓ After the recognition stage, proceed to the activation stage.
✓ C1s will bind to C4
✓ C4 cleaves to C4a and C4b
✓ C4a will be released, C4b is used
✓ C2 will cleave into C2a and C2b (released)
✓ C2a combine to c4b to form C3 convertase
✓ C3a and C3b forms
✓ C3b combines with C3 convertase to become C5 convertase
✓ After activation stage, MAC
✓ MAC will bind to C5 and cleave into C5a and C5b
✓ C5a – released
✓ Activation of C6,C7,C8,C9
✓ Formation of MAC
✓ MAC effect: hole then lead to cell lysis

Factor B
• Role is analogous to C2 of classical pathway
• Foreign cells bind to another plasma protein (factor B)
 • Binding results in the exposure of another binding site of another enzyme called Factor D

Factor D
• Factor D cleave factor B: Ba and Bb
• Bb remain bound to C3b while Ba and factor D disperse away
• Complex C3bBb is C3 convertase of alternative pathway
• Steps:
✓ C3bBb is much more efficient in cleaving C3
✓ C3b produced remains bound to C3bBb (properdin stabilizer)
✓ Will form C3bBb3b(P) (c5 convertase)
✓ C5 convertase cleaves C5 to C5a, C5b
✓ C5a - released
✓ C5b discard membrane which is 6,7,8,9
✓ Start the membrane attack complex
✓ Then C6,7,8,9 again

Properdin Factor P
• Increase half-life from 90 seconds to a few minutes
• Stabilizes C3b-Bb so it may cleave more C3, may cleave over a million C3 molecules
• Protects Bb-C3b from inactivation by complement control mechanisms.
MANNOSE-BINDING LECTIN

• Bacteria presence
• Major attack complex – an endpoint that’s why lysis happens
• MBL binds to mannose residues and certain other sugars on many pathogens
• MBL, like C1q, is a 2-to-6-headed molecule that forms a complex with 2 protease zymogens
(MASP-1 and MASP-2)
• When the MBL complex binds to a pathogen surface, MASP-2 is activated to cleave C4 and C2
• A c3 convertase is formed from C2a bound to C4b
• The MBL pathway is of importance in innate host defence mechanisms in early childhood

Activation of the lectin pathway

• 1st step is binding of Mannose Binding Lectin (MBL) to mannose residue on the surface of
microbes.
• To the MBL bound to the microbe, MBL - Associated Serine Proteases (MASP - 1 and MASP - 2)
will bind.
 • MASP 1 and 2 are similar in structure and function to Cls and
• This complex will cleave C4 and C2

Same pathway = classical and leptin

• In classical C1q,r,s
• In lectin MBL complex

Activation
• Activation of classical is ag-ab
• Activation of lectin is recognition of MBL on the surface of the pathogen
• Steps:
✓ Alternative starts in C3
✓ The cell wall of the pathogen activates C3
✓ C3 becomes C3a and C3b
✓ Factor B activates Factor D
✓ BaBb binds to C3…
✓ C5 convertase will cleave C5a and C5b
✓ C6,7,8,9

EFFECTOR FUNCTIONS OF COMPLEMENT SYSTEM

• Facilitates opsonization
• Cell lysis
• Immune complex clearance
o Inflammatory response and chemotaxis

REGULATION OF THE COMPLEMENT SYSTEM

1. C1 inhibitor
• Important regulator of the classic pathway
• Serine protease inhibitor (serpin)
• Irreversibly binds to…
✓ Properdin
✓ Factor 1
✓ Decay accelerating factor (DAF)
✓ C4B binding protein
✓ Complement receptor 1
✓ Protectin (CD59) and Vitronectin (S protein)
Consequences of Complement Deficiency
Complement Deficiency Associated Disease
C3 and Factor B Severe bacterial infections
C3b-INA, C6 and C8 Severe Neisseria infections
Deficiencies of early C components Systemic lupus erythematosus (SLE),
(C1, C4, C2) glomerulonephritis, and polymyositis
C1-inhibitor Hereditary angioedema

LABORATORY DETECTION OF COMPLEMENT ABNORMALITY

Categories
Measurement of components as antigens in serum
Measurement of functional activity

Immunologic assays of individual components

- Radial immunodiffusion and nephelometry
- Components usually measured
o C1q, C4,C3,C5,Factor B, Factor H, Factor I, C1 inhibitor
o Kits available for C3a, C4a. C5a

Test for Complements

To test the classical

Hemolytic titration assay
Lytic activity
ELISA

Test the alternative

- AH50
ELISA

Other tests
- Quantitative tests
- Test all 3 pathways using test strips coated with reagents specific for each pathway
Use complement fixation to detect antibodies, hemolysis of RBCs

Assay for Classical

- Assay that measure lysis, the end point of complement activation
- Hemolytic titration assay

Total Hemolytic Complement Assay (CH50)

- Measures ability of classical pathway and MAC to lyse sheep RBC to which Ab has been attached
 - This estimates the standard quantity of serum required as a complement sourse to produce lysis of
50% of standard quantity of sensitized sheep red cells

CH50
- Random unit defined as the quantity of complement needed for 50% red cell lysis
- Determined under standardized conditions which depend upon
- Erythrocyte and Ab conc
- Buffering Conc of medium
- Temperature

Complement hemolytic activity

- Test of classical or alternative pathway of the complement system in plasma or serum
- CH50 (classical) is based on lysis of sensibilized sheep erythrocytes in the presence of calcium
and magnesium
- Screening test when complement depletion or deficiency is suspected
- Sensitive to reduction, absence and inactivity of the component of the pathway

Principle
- Erythrocyte suspension is incubated for 30 mins with serial diluted serum or plasma at 37C. The
activation of the component system weill result in hemolysis. After incubation the samples are
centrifuged to obtain supernatant
- Free Hgb concentration in the supernatant is directly proportional to complement activity is
measured by specftrophotometer at 415 nm
- Plotting dilution factor of plasma against hemolysis degree allo the calcium of CH50 (dilution of
plasma to obtain 50% cell lysis)

Alternative Pathway
AH50 same as CH50 except magnesium chloride and thylene glycol tetraacetic acid are added to the buffer
and calcium is left out

Interpretation of lab results for complement deficiency

CH50 0 or very low

AH50 is OK
Missing C1q,C1r, C1s,C2,C4

AH50 0 or very low

CH50 is OK
Missing B or D, Properdin

AH50 and CH50 = 0 or very low

Missing C3, C5, C6, C7, C8, C9

Late components low, esp. C3, AH50, CH50

Missing factor H or factor I

(PUT PICTURE)
Complement Fixation
May serum na may Ab, may specific Ag na iadd and magbbind sa Ab
Mag aadd ng – to Ag-Ab complex
Test (put picture)

Reactive (put picture)

Serum w/ Ab
Specific Ag bind to Ab
Complement bind to Ag-Ab complex
Sensitized red cells added but no surplus complement
Intract red cells settle in pellet

Nonreactive (put picture)

Hypersensitivity

Immune system
- Provide protection against pathogens
- Distinguish self and non self
- Sometimes can cause damage
- Instead of protection, it overreacts to mechanism of self

Hypersensitive
- Over ability to detect and respond to slight changes and signals
- Any immune response that is excusively above normal
- Inappropriate immune response
- Result in host damage
- All hypersensitivity reactiomns are the consequence of adaptive immune response, specifically
caused by antibody in humoral and T cells in cellular
- Manifested in second or subsequent exposure to antigen
- based on effector mechanism infolved and time of onset

Type 1 Hypersensitivity
- IgE mediated hypersensitivity due to receptors in mast cell
- Mast cell – has IgE receptor
- Immediate hypersensitivity
- Time of onset: 2-30 minutes

Type II Hypersensitivity
- IgG or IgM recognize and react with antigen present on cells and tissues
- Complement activation due to the Ag-Ab complex formed
- Complement activation and Phagocytosis leads to cellular destruction
- Eg. Blood transfusion
- Antibody-mediated cytotoxicity
- Time of onset: 5-8 hours
Type III Hypersensitivity
- Caused by immune complexes
- Insoluble immune complexes is deposited to various body sites and leads to complement activation
(tissue damage)
- Rheumatoid arthritis
- Immune complex
- Onset: 2-8 hours

Type IV
- Caused by antigen specific effector T cells (cellular)
- Cell mediated hypersensitivity
- Nickel allergy
- Poison ivy rash
- Delayed type hypersensitivity

Type 1-3 – humoral adaptive

Type 4 – cellular

Type 1 – IgE
Type 2 – Antibody mediated
Type 3 – Immune complex
Type 4 – cell mediated

Type 1 Hypersensitivity Features

- Drug allergy
- Food allergy
- Skin allergy
- Dust allergy
- Animal allergy

Allergens – non microbial

- Pollen
- Dust
- Food
- Drugs
- Mostly protein

Development
- Genetic or environment

Atopy (atopic reaction) IgE mediated hypersensitivity

Localized
- Allergic asthma (airway)
 - Eczema (skin)
- Hay fever (nose)
- Systemic
- Whole body
- Anaphylaxis
- Venom: bee, wasp, insect
- Drugs

Treatment
- Avoidance of known allergens
- Localized atopic reactions: antihistamine
- Systemic atopic reactions: epinephrine

IgE Normal
- do not respond to allergen
- Eliminate allergen with help of IgM, IgG, IgA
- No clinical symptom

IgE Atopic Person

- Respond to allergen
- Produce IgE (hallmark)
- Show clinical symptoms

Component of the Immune System

- Mast cell
- IgE
- Basophil
- Eosinophil;
- Th2
- IL4, IL5, IL13
- Cytokines and chemokines

Type I Hypersensitivity Mechanism

Hypersenstiivity reactions
Sensitization stage
- First encounter to allergen
- Dendritic cells present allergen to naïve T cell to produce IgE and effector cells (mast cell,
eosinophil, basophil)
- IgE binds to mast cells because of its receptor

Effector stage

Steps
1. Exposure to allergen
2. Dendritic cell present to naïve T cell
3. Naïve T produce IL4 which has an effect to Th2 which will produce IL4
4. B cell proliferates into plasma cells and produce IgE
 5. There is also production of cytokines which has effector functions for basophil, eosinophil, mast cell
then the activation of effector cells of allergy

2nd Exposure to Antigen

- Antigen attached to antibody (IgE-Ab attached to mast cell
- Preformed
- Histamine is released which leads to nerve stimulation, then vasodilation (redness) then fluid
leakage (swelling)
- Mast cell will produce granules

Allergic Cascade
- Degranulated mast cell will release histamine which leads to nerve stimulation, then vasodilation
(redness) then fluid leakage (swelling)

Sensitization Stage (1st exposure)

- B cell produce IgE and attach to mast cell

Effector Stage (2nd exposure)

- Mast cell with IgE is exposed to allergen then the antigen (allergen) attaches to IgE attached to the
mast cell

Type II
- Ab mediated … (CONTINUE)
- Drug has self Ag then its modified and penicillin attaches and becomes an alloantigen – non self
Ag from members of same species

Characteristic Features
- IgG or IgM + Ag or hapten on membrane
- Injury and dysfunction of target cells then cell lysis

Antibodies Involved (IgG and IgM)

- Autoantibodies – Produced due to failure in self tolerance mechanism
- Alloantibodies – produced in Rh(-) mother on coming in contact with Rh(+) cells

Landsteiner Rule
- If an antigen is absent, the corresponding antibody is absent
- Forward – test Ag
- Reverse – test Ab
- Anti D – only antibody to cross the placenta

Other Components
- Neutrophil
- Eosinophil
- Natural killr
- Macrophage
- Complement protein
Eg. of Type II Hypersensitivity
- Hemolytic anemia
- Blood Transfusion
o Type A (A Ag) nasalinan ka ng Type B (Anti-A) therefore causes lysis
- Rh incompatibilioty
- Tissue transplantation rejection

Consequences of Ag-Ab Interaction

- Ag are found on cell surface
- IgM, IgG

Autoimmune hemolytic anemia and type II drug reaction (PUT PICTURE)

Drug Induced Hemolytic Anemia (PUT PICTURE)

- Certain antibiotics can be absorbed

HAR hyperacute graft rejection

- Destruction of graft immediately after transplantation
- Pre existing antibodies in the receipients circulation
- Recognize alloantigens present on the endothelial cells…

Transplanted kidney
- Have circulating alloantigen
- Breaks the graft transplanted

Good Pasteur syndrome

- Antigens form antibodies attach basement cell membrane affects kidney …

B. Cytotoxic Antibodies to Tissue Components

- cell injury may be brought about by autoantibodies reacting with some components of tissue cells

Mechanism
- Complement activation
- opsonization OF opsonized phagocytosis
- ADCC
- Classical pathway

Complement activation
- Hemolytic anemia
- Transfusion…

ADCC
- Ab has tumor cells
- Activate NK cells
- It binds and release granules
- Apoptosis
Test for Type II
Direct Coombs Test/Direct antiglobulin test (PUT PICTURE)
Polyspecifc AHG mix: IgG complements (C3b)
Monospecific AHG: Anti IgG, anti C3b and anti C3d

Type III Hypersensitivity

- Immune complex mediated hypersensitivity
- Ag-Ab complex
- Deposit at various sites of the body
- What removes Ag-Ab complex? Complement

Features
- Exogenous antigens – proteins produced by pathogens
- Endogenous antigens – … (I DON’T KNOW)
- Ab involved – mostly IgG
- Mast cells, neutrophils, …

Localized
- Limited to area of immune complex first deposited
- Pain, redness, swelling

Systemic
- Autoantibodies are formed against self antigens

RA (??)
- Autoantibodiues are formed against IgG molecules of the individual
- Joints

Mechanism
- Immune complex formation is normal – complement activation
- Soluble immune complex
- Cleared by phagocytes
- Trigger when immune complex attach certain size
- Cannot be phagocytised
- Insoluble
- Start depositing in the body
- Kidney (glomerulonephritis), joint (RA), vasculitis (?)

Ag-Ab
Immune complex
Activate complement
Remove complex

Type IV Hypersenstivitiy Reaction

- Th, Tc
 - Neutrophil, Macrophage, mast cell (?)
- Cell mediated

Ag triggering type IV hypersensitivity

- Foreign agents that alter self antigen
- Auto

Reaction develops in 2 stages

A. Sensitization Stage
- Primaru contact
- T cells are sensitized and …
B. Effector Stage

(Image: sensitization phase and Effector Phase)

Please read and correlate with the mechanism of hypersentivity:

- Transfusion reaction
- HDN, AIHA
- Arthus reaction, serum sickness
- Hypersinsitivity pneumonitis

[AUTOIMMUNE]

Autoimmune DSE- conditions in which damafe to orfgans or tissues fro, the presence of autoantibody
Review:
TOLERANCE:Is the lack of immune response to self antigen and is initiated
a. Central Tolerance- coccurs during lymphocyte development and operated in thymus and bone
marrow where there is deletion of the lymphocytes that recognize self antigens. This process is
most active in fetal life, but continues as immature lymphocytes
b. Peripheral- some auto reactive cells escap the peripheral circulation, where they are suppressed
by regulatory T cells
Thymus
- Positive selection
- Negative selection

Causes of immunity
- Genes
- Immune regulation
- Environment

Other causes
- Sequestered antigens or hidden antigens: Abtigens in secluded places are not accessib;e to the
immune system. Ex. Lens Ag. Sem Ag

Autoimmune DSE
1. He,olutics
2. Sytemic
 3. Licalized

A. Systemic
- Rhumatod arthritis

B. Organ Specific
- Multiple sclerosos, myasthenia gravis, Cheronic thyroiditis

C. Systemuc LupusnErthematous
- Chronic SYSTEMIC INFLAMMATORY DSE
- Characterized by:
- Alyernating increasein severity amd remission
- Peak age of onsetr 20 and 40
- Eiology: idiopaythis
- Genertic, environmental, hoemones can be a predisposing factor
- Infection
- Antibiotics (sulfoinamides and peniciilin derivatives)
- Ultraviolet light
- Extreme stress
- Combination of thos factor
- Hormoinal influence: estrogen
- Genetic predispositoio: Known to occus within familit but no identofioed genes assoc eith lupus
- Envi: UV light → DNAR → Thymine fimer→ Dormatuon of anti DNA
- Can be acute or chronic inflammation
- Signs: Fever, weight loss, joint pain, argtritis, erythaomatous, maculopapular rash (Buttterrlffy)
- Compelment mediated injuti

Pathogenesis
- Tlumphocyre, blymphocyre, dendri cells
- Loss of tolerance to self antigen, depositiin, of immune complexes in ytiossues, multiorgan
involvement
- Circulation immune comples
- Antibodies to hosty antigen(nuc;ear antigen DNA) typical antibodies produced
- Antonuclear antibodt (ANA)
- Heterogenous group of antibodies

Diagnostic Evaluation
- Histologux chanfes:
1. Acute vasculitis
2. Persistent inflammation

- Hemostatic Testing
a. Lupius
b. Prolonged ptothrombin time

- Serological
a. High level of anti DNA
 b. Reduce complement leves

- Antoibodies
a. Non specific elevation of immyunoglbulin

ANTINUCKLEAR ANTIBODIES
- Hetyerogenous group circulatin igm, igg and igg
- React with whole nuckleaus or nuclear components (Histone, prirteins, DNA) in host tissie

LABIRATORY DIAGNOSIS
- NA: Screening test 95 % of SLE patienty have ANA so absence of ANA can rule out SLE but
presence of ANA cannot conform SLE
- Immunofluorescence
a. Extremely sensitive
b. Most widely used technique for ANA
- Indirect immunofluorescent test for antinuclear antobidy
a. Based on the use of flurosceuin antoglobulin

Stage of immunofluorescenxe for the detection of antinucleae antiboduies.

Hep-2 cells (rat or lmouse lives or kidney contains substrate)
1. And then incubayted with a persons blood serum
2. Of the serum vontains antibodies, they will bind to antigens within the Hep-2-cell nucleus
3. These antobosied can be visulaizerd by subsequent incubvation with antin human antibodies
conjugated to a flurescent molecule

Immunological Criteria
- dsDNA ab most specific for SLE
- Diagnostoc in combibnation with low levels of C#
- Antinuvclear antibodies (ANA) level above;aboratorory referemce tange
- Anti-double stranded DNA (dsDNA) antibody level above laboratory reference range [or >2 fols the
reference range if ested by ELISA
- Anti-smith: presence of antibody to smith nuclear antigen

Nuclear antigen (Nucleus)

1. ANA
2. Anti DNA
3. Anti-Sm
4. Anti RNO
5. Anti Ro
6. Anti La

Tests:
1. CBC
2. Urinalysis
3. ESR
4. RF
 5. Skin Biopsy
6. Kidney Biopsy

EFFECT OF LABORATORY TESTS WITH INCREASE LUPUS ACTIVITY

↑CRP
↑ESR
↑Anti DNA

[RHEUMATOID ARHTRITIS]
- DSE of the joints
- Caused by the autoantibofy of IgM type, called as Rf
- Increases with age
- More common in female than female
- 3 distinct syages

[AUTOIMMUNE-PART 2]
- Autoimmiune hemolytic anemia
- Warm reactive autoantibodies (most common)

WARM:
- Antibodies reactive at 37C
- RBC coated with Igg and complement
- Elution
- IgG binds to RBC surface antigens
- This drives monocytes & macrophages to grav and pick off portions of RBC memnvrane
COLD
- Acute from secondary to mycoplasma pneumonia infection
- Chronic form seen in older patients
- In cold temp, Igm binds topolysaccharide region og glycoproteins on RBC surfacr
- This triggers complement to lyse RBC

PAROXYSMAL COLD HEMAGLUBINURIA

- Autiantibody id IgG

DRUG INDUCED
- Drug
LABORATORY DETECTION OF COMPLEMENT ABNORMALITY
Categories
• Measurement of components as antigens in serum
• Measurement of functional activity
• Immunologic assays of individual components
• Radial immunodiffusion and nephelometry
• Components usually measured: C1q, C4, C3, C5, Factor B, Factor H, Factor I, C1 inhibitor
• Kits available for C3a, C4a, C5a
Test for Complements
• To test the classical
• Hemolytic titration assay
• Lytic activity
• ELISA
• Test the alternative
• AH50
• ELISA
• Other tests
• Quantitative tests
• Test all 3 pathways using test strips coated with reagents specific for each pathway
• Use complement fixation to detect antibodies, hemolysis of RBCs
Assay for Classical
• Assay that measure lysis, the end point of complement activation
• Hemolytic titration assay

Total Hemolytic Complement Assay (CH50)

• Measures ability of classical pathway and MAC to lyse sheep RBC to which Ab has been attached
• This estimates the standard quantity of serum required as a complement source to produce lysis of
50% of standard quantity of sensitized sheep red cells

CH50
• Random unit defined as the quantity of complement needed for 50% red cell lysis
• Determined under standardized conditions which depend upon
• Erythrocyte and Ab conc
• Buffering Conc of medium
• Temperature

Complement hemolytic activity

• Test of classical or alternative pathway of the complement system in plasma or serum
• CH50 (classical) is based on lysis of sensitized sheep erythrocytes in the presence of calcium and
magnesium
• Screening test when complement depletion or deficiency is suspected
• Sensitive to reduction, absence and inactivity of the component of the pathway

Principle
• Erythrocyte suspension is incubated for 30 mins with serial diluted serum or plasma at 37C. The
activation of the complement system will result in hemolysis. After incubation the samples are
centrifuged to obtain supernatant
• Free Hgb concentration in the supernatant is directly proportional to complement activity is
measured by spectrophotometer at 415 nm
• Plotting dilution factor of plasma against hemolysis degree allow the calculation of CH50 (dilution of
plasma to obtain 50% cell lysis)

Alternative Pathway AH50 same as CH50 except magnesium chloride and ethylene glycol tetraacetic acid
are added to the buffer and calcium is left out
Interpretation of lab results for complement deficiency
1.CH50 0 or very low
• AH50 is OK
• Missing C1q, C1r, C1s, C2, C4
2.AH50 0 or very low
• CH50 is OK
• Missing B or D, Properdin
3.AH50 and CH50 = 0 or very low
• Missing C3, C5, C6, C7, C8, C9
4.Late components low, esp. C3, AH50, CH50
• Missing factor H or factor I

Complement Fixation
• May serum na may Ab, may specific Ag na iadd and magbind sa Ab
• Mag aadd ng – to Ag-Ab complex
• Test

Reactive
• Serum w/ Ab
• Specific Ag bind to Ab
• Complement bind to Ag-Ab complex
• Sensitized red cells added but no surplus complement
• Intract red cells settle in pellet

Nonreactive
•

HYPERSENSITIVITY
Immune system
• Provide protection against pathogens
• Distinguish self and non-self
• Sometimes can cause damage
• Instead of protection, it overreacts to mechanism of self

Hypersensitive
• Over ability to detect and respond to slight changes and signals
• Any immune response that is exclusively above normal
• Inappropriate immune response
• Result in host damage
• All hypersensitivity reactions are the consequence of adaptive immune response, specifically
caused by antibody in humoral and T cells in cellular
• Manifested in second or subsequent exposure to antigen
• Based on effector mechanism involved and time of onset

Type 1 Hypersensitivity
• IgE mediated hypersensitivity due to receptors in mast cell
• Mast cell – has IgE receptor
 • Immediate hypersensitivity
• Time of onset: 2-30 minutes

Type II Hypersensitivity
• IgG or IgM recognize and react with antigen present on cells and tissues
• Complement activation due to the Ag-Ab complex formed
• Complement activation and Phagocytosis leads to cellular destruction
• Eg. Blood transfusion
• Antibody-mediated cytotoxicity
• Time of onset: 5-8 hours

Type III Hypersensitivity

• Caused by immune complexes
• Insoluble immune complexes is deposited to various body sites and leads to complement activation
(tissue damage)
• Rheumatoid arthritis
• Immune complex
• Onset: 2-8 hours

Type IV Hypersensitivity
• Caused by antigen specific effector T cells (cellular)
• Cell mediated hypersensitivity
• Nickel allergy
• Poison ivy rash
• Delayed type hypersensitivity

Type 1-3 – humoral adaptive Type 4 – cellular

Type 1 – IgE Type 2 – Antibody mediated Type 3 – Immune complex Type 4 – cell mediated

Type 1 Hypersensitivity Features

• Drug allergy
• Food allergy
• Skin allergy
• Dust allergy
• Animal allergy

Allergens – non-microbial
• Pollen
• Dust
• Food
• Drugs
• Mostly protein

Development
• Genetic or environment
• Atopy (atopic reaction) IgE mediated hypersensitivity
Localized
• Allergic asthma (airway)
• Eczema (skin)
• Hay fever (nose)
• Systemic
• Whole body
• Anaphylaxis
• Venom: bee, wasp, insect
• Drugs

Treatment
• Avoidance of known allergens
• Localized atopic reactions: antihistamine
• Systemic atopic reactions: epinephrine

IgE Normal
• do not respond to allergen
• Eliminate allergen with help of IgM, IgG, IgA
• No clinical symptom

IgE Atopic Person

• Respond to allergen
• Produce IgE (hallmark)
• Show clinical symptoms

Component of the Immune System

• Mast cell
• IgE
• Basophil
• Eosinophil;
• Th2
• IL4, IL5, IL13
• Cytokines and chemokines

Type I Hypersensitivity Mechanism Hypersensitivity reactions Sensitization stage

• First encounter to allergen
• Dendritic cells present allergen to naïve T cell to produce IgE and effector cells (mast cell,
eosinophil, basophil)
• IgE binds to mast cells because of its receptor

Effector stage
Steps
1. Exposure to allergen
2. Dendritic cell present to naïve T cell
3. Naïve T produce IL4 which has an effect to Th2 which will produce IL4
4. B cell proliferates into plasma cells and produce IgE
 5. There is also production of cytokines which has effector functions for basophil, eosinophil, mast cell
then the activation of effector cells of allergy

2nd Exposure to Antigen

• Antigen attached to antibody (IgE-Ab attached to mast cell
• Preformed
• Histamine is released which leads to nerve stimulation, then vasodilation (redness) then fluid
leakage (swelling)
• Mast cell will produce granules

Allergic Cascade
• Degranulated mast cell will release histamine which leads to nerve stimulation, then vasodilation
(redness) then fluid leakage (swelling)

Sensitization Stage (1st exposure)

• B cell produce IgE and attach to mast cell

Effector Stage (2nd exposure)

• Mast cell with IgE is exposed to allergen then the antigen (allergen) attaches to IgE attached to the
mast cell

Type II
• Ab mediated … (CONTINUE)
• Drug has self Ag then its modified and penicillin attaches and becomes an alloantigen – non self
Ag from members of same species

Characteristic Features
• IgG or IgM + Ag or hapten on membrane
• Injury and dysfunction of target cells then cell lysis

Antibodies Involved (IgG and IgM)

• Autoantibodies – Produced due to failure in self tolerance mechanism
• Alloantibodies – produced in Rh(-) mother on coming in contact with Rh(+) cells

Landsteiner Rule
• If an antigen is absent, the corresponding antibody is absent
• Forward – test Ag
• Reverse – test Ab
• Anti D – only antibody to cross the placenta
Other Components
• Neutrophil
• Eosinophil
• Natural killer
• Macrophage
• Complement protein
• Eg. of Type II Hypersensitivity
• Hemolytic anemia
 • Blood Transfusion
• Type A (A Ag) nasalinan ka ng Type B (Anti-A) therefore causes lysis
• Rh incompatibility
• Tissue transplantation rejection

Consequences of Ag-Ab Interaction

• Ag are found on cell surface
• IgM, IgG

Autoimmune hemolytic anemia and type II drug reaction

• Drug Induced Hemolytic Anemia
• Certain antibiotics can be absorbed

HAR hyperacute graft rejection

• Destruction of graft immediately after transplantation
• Pre existing antibodies in the recipients circulation
• Recognize alloantigens present on the endothelial cells…

Transplanted kidney
• Have circulating alloantigen
• Breaks the graft transplanted

Good Pasteur syndrome

• Antigens form antibodies attach basement cell membrane affects kidney …

B. Cytotoxic Antibodies to Tissue Components

• Cell injury may be brought about by autoantibodies reacting with some components of tissue cells

Mechanism
• Complement activation
• Opsonization OF opsonized phagocytosis
• ADCC
• Classical pathway

Complement activation
• Hemolytic anemia
• Transfusion…

ADCC
• Ab has tumor cells
• Activate NK cells
• It binds and release granules
• Apoptosis

Test for Type II Direct Coombs Test/Direct antiglobulin test Polyspecific AHG mix: IgG complements (C3b)
Monospecific AHG: Anti IgG, anti C3b and anti C3d
Type III Hypersensitivity
• Immune complex mediated hypersensitivity
• Ag-Ab complex
• Deposit at various sites of the body
• What removes Ag-Ab complex? Complement

Features
• Exogenous antigens – proteins produced by pathogens
• Endogenous antigens – … (I DON’T KNOW)
• Ab involved – mostly IgG
• Mast cells, neutrophils, …

Localized
• Limited to area of immune complex first deposited
• Pain, redness, swelling

Systemic
• Autoantibodies are formed against self antigens

RA (??)
• Autoantibodies are formed against IgG molecules of the individual
• Joints

Mechanism
• Immune complex formation is normal – complement activation
• Soluble immune complex
• Cleared by phagocytes
• Trigger when immune complex attach certain size
• Cannot be phagocytized
• Insoluble
• Start depositing in the body
• Kidney (glomerulonephritis), joint (RA), vasculitis (?)

Ag-Ab Immune complex Activate complement Remove complex

Type IV Hypersensitivity Reaction
• Th, Tc
• Neutrophil, Macrophage, mast cell (?)
• Cell mediated

Ag triggering type IV hypersensitivity

• Foreign agents that alter self antigen
• Auto

Reaction develops in 2 stages A. Sensitization Stage

• Primary contact
• T cells are sensitized and … B. Effector Stage
(Image: sensitization phase and Effector Phase)
Please read and correlate with the mechanism of hypersensitivity:
• Transfusion reaction
• HDN, AIHA
• Arthus reaction, serum sickness
• Hypersensitivity pneumonitis

AUTOIMMUNE DISEASES
- Conditions in which organ or tissue damage results from the presences of autoantibody or
autoreactive cells

Peripheral Tolerance
- tolerance induced in mature lymphocytes from the outside of the primary lymphoid organs

Central Tolerance
- destruction of self-reactive lymphocytes in the primary lymphoid organs

Organ Specific
- Multiple sclerosis
- Myasthenia gravis
- Chronic thyroiditis
- Graves disease
- Type 1 diabetes
- Pernicious anemia
- Idiopathic thrombocytopenia
- Guillain Barre syndrome

Systemic Lupus Erythematosus

- Chronic systemic inflammatory disease
- Characteristics
o Alternating increase in
o Peak age of onset 20 and 40
o Common to women than
o African American and Hispanics than in Caucasians

- Positive Selection – Cells that recognize self MHC or recognized and bind to peptide + MHC
molecule selected to grow
- Negative Selection – Cells that recognize self peptides are autoreactive cells and undergo cell
death because they are harmful to the host
- Cells that pass both, enter circulation

Other causes
Sequestered or hidden antigens
- Antigens in secluded places are not accessible to the immune system
Neoantigen
 - Altered or modified antigen
Physical – irradiation
Chemical – drugs
Microbial agent – viruses

Central tolerance
- Occurs during lymphocyte development
- Operates in thymus and bone marrow where there is deletion of the lymphocyte that recognize self
antigens
- This process is more active in fetal life but continues as immature lymphocytes develop throughout
life

Systemic (Collagen or Vascular)

- Rheumatoid arthritis
- Systemic lupus erythematosus
- Good Pasture syndrome

Diagnostic Evaluation
- Histologic changes
o Acute vasculitis
o Persistent inflammation
- Hematological
o Moderate anemia
o Coating of erythrocytes (AHG test)
o Lymphocytopenia
o Thrombocytopenia

Hormonal influence
- Estrogen

Genetic predisposition
- Known… within families but no identified… with lupus
Environmental: UV light
Formation of anti-DNA

Antibodies to host antigens (nuclear antigens DNA) typical antibodies produced

Antinuclear antibodies
- Heterogenous group of antibodies produced against variety of antigens within the
- Can be found in disease other (rheumatic or non rheumatic)

Serological
- High level of anti DNA
- Reduced complement levels
- Presence of complement breakdown of C3 (C3d and C3c)
- Cryoglobulin level correlate… SLE
- Can be acute or chronic inflammation
- Signs: fever, weight loss, joint pain, arthritis, erythematous, maculopapular rash
 - Complement mediated… system due to high level of imm… deposited in the kidneys

Pathogenesis
- T and B lymphocyte, Dendritic cells
- Loss of tolerance to self antigens
- Circulating immune complexes – hallmark of SLE

Hemostatic testing
- Lupus anticoagulant, antiphospholipid seen in SLE
- Prolonged prothrombin time and part … thromboplastin time

Etiology: idiopathic
Genetic, environmental hormones – predisposing factor

- Infection
- Antibiotics (sulfonamides and p
- UV light
- Extreme stress
- Combination of

Serological
- High anti DNA level
- Reduced complement levels
- Presence of complement breakdown (C3d and C3c)
- Cryoglobulin level correlated… of SLE

Laboratory Diagnosis
- ANA: screening test 95% of SLE patient have ANA so absence of ANA can rule out SLE but
presence of ANA cannot confirm SLE
- Immunofluorescence
o Extremely sensitive
o Most widely used technique for ANA
- Indirect immunofluorescent test for anti…
o Based on the use of fluorescein

Antinuclear antibodies
- Heterogenous group circulating IgM, IgG, IgA
- React with whole nucleus or nuclear (histone, proteins, DNA) in host
- Assay not specific for SLE as they

Antibodies
- Non specific elevation of IgG and IgM
- IgA deficiency
- ANA
dsDNA ab – most specific for SLE
Diagnostic in combination with low C3 levels
Stages of Immunofluorescence for the detection of antinuclear antibodies
Hep-2 cells (rat or mouse liver or kidney conta
1. and then incubated with person’s blood serum
2. if the serum contains Ab, they … within the Hep-2 cell nucleus
3. These antibodies can be visu…

3 Stages
- Synovitis initiation
- Subsequent immunologic events
- Transition of inflammatory reacti

Autoimmune hemolytic anemia

- Warm reactive autoantibodies – most common
- Cold react
- Paroxysmal cold hemoglobinuria
- Drug induced hemolysis

Rheumatoid arthritis
- Disease of the joints
- Caused by auto antibody of IgM type – rheumatoid factors
- Synovial fluid of these patients contain increased T cells and macrophages
- Marked by inflammatory changes in the synovial membrane
- Later stages – deformity develops
- Increases with age
- More common in female
- Chronic
- Multi systemic
- Autoimmune and progressive inflammatory disease
- Associated with MHC Class II genes
- DR4 alles

Idiopathic Thrombocytopenic Purpura

- IS destroys blood platelets which are needed for blood to clot
- Major categories
o Decreased platelet production
o Platelet distribution disordered
o Increased platelet destruction or use

Paroxysmal Cold Hemoglobinuria

- Autoantibody: IgG (Donath Landsteiner)
- Produces complement C3 and C4 to bind irreversibly to the RBC
- Elution detection
- Donath Landsteiner test

Drug Induced Hormones

- DAT
- Stages
 o Drug absorption
o Immune complexing
o Membrane modification
o Autoantibody formation

Hashimoto’s Thyroiditis
- Caused by throid gland inflammation
- Most common thyroid disease
- Destruction of thyroid
- Caused by auto Ab of IgG and IgM

Cold Agglutination Disease

- In cold temperatures, IgM binds to polysaccharide region of glycoproteins on RBC surface
- This triggers complement system to lyse RBC -> Intravascular hemolysis

- If complement system fails to form membrane complex (when trigger is insufficient),

- Opsonization enhances RBC phagocytosis -> Extravascular hemolysis

Direct Coomb’s Test (Direct Antiglobulin Test)

- Determine whether RBC binding autoantibody (IgG) OR C3 is bound to Ag on RBC membranes
- Coomb’s reagent is added to washed RNC’s f
- If IgG or C3 is bound to RBC membrane, agglutination happens – positive result
Warm Autoimmune Hemolytic Anemia
- IgG binds to RBC surface antigens
- Drives monocytes and macrophages to grab and pick off portions of RBC membrane
- RBCs become spherocytes
- Destructed in spleen -> Extravascular hemolysis

Warm Autoimmune Hemolytic Anemia

- Ab reactive at 37 C
- RBC coated with IgG and complement
- Elution (demonstration)

Cold Autoimmune Anemia

- Acute form secondary to mycoplasma
- Chronic form seen in older patients
- Cold reactive Ig autoantibody
- Complement is the only globulin

Drug Induced
- Drug binds to and reacts with red cell surface proteins
- Antibodies recognize altered proteins, as foreign
- Antibodies bind to altered protein and lead to hemolysis
- Drugs: Quinidine, sulfonamide derivatives: interaction of platelet antigen with drug antibodies
- Bacterial sepsis: increased destruction of platelet due to attachement of plately to bacterial AG-Ab
complexes
 - Post transfusion purpura
- Increased platelet destruction caused by antiplatelet antibodies -> antibodies directed against
platelet membrane antigens such as GPIIb/IIIA -> the platelets coated with immune complexes
bind to Fc portion of macrophages in spleen and other RES and are removed
- Lack of compensatory response by megakaryocytes due to suppressive effect of antiplatelet
antibodies
- A combination of increased platelet destruction lead to ineffective megakaryopoiesis

Diabetes mellitus type 1

- Insulin dependent
- Inflammatory autoimmune disease of the pancreas
- Lack of insulin
- Insulin
o Produced in the pancreas by the beta cells of the islets of Langerhans
o For glucose to get into and used for energy production
o After eating, glucose level rises, and leads to insulin released
- Damaged beta cells of the Islets of Langerhands
- In type 1 DM, effector T cell recognizes peptides from Beta cell specific protein and kills the beta
cell
- Glucagon and somatostatin are still produced by the Beta and delta cells but not insulin can be
made
- Alpha cell – glucagon
- Beta cell – insulin
- Delta cell – somatostatin

Myasthenia gravis
- Caused by defect in transmission of nerve impulses to muscles
- Occurs when normal communication between nerve and muscle is interrupted at the
neuromuscular junction – place where nerve connect with muscles
- When impulses travel down, nerve endings release acetylcholine
- Results in skeletal muscle weakness and fatigue

Pathophysiology
1. Acquired immunologic abnormality
- autoantibodies against nicotinic Ach receptors in the muscles (80-90%)
- MuSK (muscle specific receptor tyrosine kinase antibodies)
2. Structural abnormality
3. Thymus

Multiple sclerosis
- Degeneration of nerves CNS (brain and spinal cord)
- Myelin disappear due to inflammation

Addison’s Disease
- Aka primary adrenocortical insufficiency
- Presence of autoantibodies directed against 21-hydroxylase, a key regulator of mineralocorticoid
and glucocorticoid synthesis
Grave’s Disease
- Thyroid hormone production is regulated by TSH
- Binding of TSH to a receptor on thyroid cells activate adenylate cyclase and stimulates the
synthesis of 2 thyroid hormones: thyroxine and triiodothyronine

Celiac disease
- Inherited condition
- Prevents small intestine from absorbing nutrients, causing malnutrition

HIV 1 vs HIV 2
- HIV 2
o Lower transmissibility
o Develops slowly
- Mother to child transmission – rare in HIV 2
- HIV 1 – more common

Multiple sclerosis (MS) and Guillain-Barré syndrome (GBS)

- diseases of the nervous system. They're not the same, but they do have a lot of similarities.
- Both MS and GBS are autoimmune diseases.
- This means they cause your body's immune system to attack its own tissues. They both start when
the immune system attacks and damages something called myelin. That's a layer of insulation that
surrounds nerves
- Each condition affects a different part of your nervous
- MS damages the central nervous system. That's spinal cord.
- GBS damages the peripheral nervous system

Guillain - Barre Syndrome

- IS attacks the nerves that connect your brain and spinal cord with the rest of your body.
- Damage to the nerves makes it hard for them to transmit signals, as a result the muscle have
trouble responding to the brain