SERO_SEM

Hypersensitivity

Heightened state of immune responsiveness/exaggerated immune response against a harmless antigen

time course

T cells

• Different types of hypersensitivity reactions are distinguished by:

  • Their _ _

  • Whether antibodies or _ _ are the principle immune elements involved

IgE

IgG or IgM

IgG or IgM

T cells

	Type I	Type II	Type III	Type IV
Immune Mediators

Anaphylactic

Antibody-mediated cytotoxic

Complex- mediated

Cell-mediated or delayed type

	Type I	Type II	Type III	Type IV
Synonym

Immediate

Immediate

Immediate

Delayed

	Type I	Type II	Type III	Type IV
Timing

No

Yes

Yes

No

	Type I	Type II	Type III	Type IV
Complement Involvement

Autoimmune Diseases

Conditions in which damage to organs or tissues results from the presence of autoantibodies or autoreactive cells

• Decreased Treg/Tsuppressor cells

• Increased autoantibodies

• Increased Ag-Ab complexes

• Decreased complement due to consumption of complement proteins

General Signs of Autoimmune Diseases

(4)

Self-tolerance

Autoimmune Diseases

Ability of the immune system to accept self-antigen and not initiate a response against them

Central

Peripheral

Autoimmune Diseases

Types of self-tolerance

Molecular Mimicry

Individual bacterial or viral agents contain antigens that closely resemble self-antigens

Bystander Effect

Molecular Mimicry

Microorganism can induce a local inflammatory response that recruits leukocytes and stimulates APCs to release cytokines that activates T Cells

Superantigens

Molecular Mimicry

Ability to bind to both MHC Class II and TCRs, regardless of the antigen specificity

Systemic Lupus Erythematosus (SLE)

Clinical Types

Systemic

Immune complex disease characterized by overproduction of autoantibodies

butterfly (malar) rash

Clinical Types

Systemic

Systemic Lupus Erythematosus (SLE)

Manifests itself by skin lesions ?

C3

anti-nuclear antibodies (ANA) (non specific)

LE cell

Clinical Types

Systemic

Systemic Lupus Erythematosus (SLE)

• Laboratory Diagnosis

  • Complement Proteins (?)

  • Presence of ?

  • ? – PMN leukocyte with ingested LE body, often in rosette formation

Antinuclear Antibodies (ANA)

Clinical Types

Systemic

Systemic Lupus Erythematosus (SLE)

• Autoantibodies that are directed against antigens in the nuclei of mammalian cells

• Targets: Double-stranded (ds) and single-stranded (ss) DNA (deoxyribonucleic acid), histones, nucleosomes (DNA histone complexes), centromere proteins, and extractable nuclear antigens (ENAs)

Procainamide

Hydralazine

Chlorpromazine

Isoniazid

Quinidine

Clinical Types

Systemic

Systemic Lupus Erythematosus (SLE)

• Drugs associated to Lupus: (5)

Anti-dsDNA

_

dsDNA

Homogeneous

SLE

Anti-ssDNA

_

Related to purines and pyrimidines

Not detected on routine screen

SLE, many other diseases

Antihistone

_

Different classes of histones

Homogeneous

Drug-induced SLE, other diseases

Anti-DNP

_

DNA-histone complex (nucleosomes)

Homogeneous

SLE, drug-induced SLE

Anti-Sm

_

Extractable nuclear antigen (uridine-rich RNA component)

Coarse speckled

Diagnostic for SLE

Anti-RNP

_

Proteins complexed with small nuclear RNA

Coarse speckled

SLE, mixed connective tissue diseases

Anti-SS-A/Ro

_

Proteins complexed to RNA

Finely speckled

SLE, Sjögren’s syndrome, others

Anti-SS-B/La

_

Phosphoprotein complexed to RNA polymerase

Finely speckled

SLE, Sjögren’s syndrome, others

Antinucleolar

_

RNA polymerase, fibrillarin, PM-1

Prominent staining of nucleoli (can be smooth, clumpy, or speckled)

SLE, systemic sclerosis

Anti-Scl-70

_

DNA topoisomerase I

Compound pattern with speckling

Systemic sclerosis, scleroderma

Anti-Jo-1

_

Histidyl-tRNA synthetase

Fine cytoplasmic speckling

Polymyositis

Autoantibody–Anti-Centromere

_

Characteristics of Antigen-Antigens in the chromosome centromeres

Immunofluorescent pattern-discrete speckled

CREST syndrome

Calcinosis

Raynaud’s phenomenon

Esophageal dysmotility

Sclerodactyly

Telangiectasia

CREST Syndrome

Fluorescent Antinuclear Antibody Test

Methods of Detection

• Most widely used and accepted test

• Human epithelial cell line, Hep-2, is the standard substrate.

• Fluorescent Staining Pattern

Indirect immunofluorescence (IIF)

Methods of Detection

Fluorescent Antinuclear Antibody Test

• Principle

Homogenous

Peripheral

Speckled

Nucleolar

Centromere

Methods of Detection

Fluorescent Antinuclear Antibody Test

Pattern	Description
	Uniform staining of the entire nucleus
	Diffuse staining is throughout the nucleus, but greater intensity around the outer circle surrounding the nucleus
	Discrete, fluorescent specks throughout the nucleus
	Prominent staining of the nucleoli
	Numerous discrete speckles are seen

Microsphere Multiplex Immunoassay (MIA)

Methods of Detection

• Microtiter plate well containing a suspension of polystyrene microspheres that are coated with individual nuclear antigens or with a HEp-2 extract

• Antibodies in the patient serum will bind only to the beads containing their specified antigens

• Phycoerythrinlabeled anti-human IgG is added

• Bead suspension is analyzed for fluorescence by a flow cytometer that has two lasers, one that identifies each bead and another that detects the amount of fluorescent conjugate attached

Immunofluorescence Using Crithidia luciliae

Methods of Detection

• Used to detect antibodies to double-stranded DNA (anti-dsDNA antibodies)

  Trypanosome has a circular organelle called a kinetoplast that is composed mainly of dsDNA

Ouchterlony Test

Methods of Detection

• Used to determine the immunologic specificity of a positive FANA test

Antiphospholipid Antibodies

Antibodies that bind to phospholipids alone or phospholipids complexed with protein

Lupus Anticoagulant

Produces a prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT)

Extractable nuclear antigens

represent a family of small nuclear proteins that are associated with uridine-rich RNA

anti-Sm antibody

The ? is specific for lupus, because it is not found in other autoimmune diseases

Double-stranded DNA (ds-DNA) antibodies

are the most specific for SLE, the presence of these antibodies is considered diagnostic for SLE

Rheumatoid Arthritis

• Chronic, symmetric, and erosive arthritis of the peripheral joints

• Women are three times as likely to be affected as men

• Progresses to joint deformity and disability

Citrulline

Rheumatoid Arthritis

Generated when enzyme peptidyl arginine deaminase (PAD) modifies the amino acid arginine by replacing an NH2 group with a neutral oxygen

Rheumatoid factor

Rheumatoid Arthritis

Group of immunoglobulins that interacts specifically with the Fc portion of IgG molecules

Anti-cyclic citrullinated peptide (anti-CCP) antibodies

Rheumatoid Arthritis

= specific marker

Sheep Cell Agglutination Test

Latex Fixation Test

Sensitized Alligator Erythrocytes test

Bentonite Flocculation Test

Rheumatoid Arthritis

Laboratory Tests (4)

Sjögren’s syndrome

Other Systemic Autoimmune Rheumatic Disease (SARDS)

characterized by chronic inflammation of the exocrine glands, most notably the ocular and salivary glands

Scleroderma (SSc)

Other Systemic Autoimmune Rheumatic Disease (SARDS)

rare SARD that is characterized by excessive fibrosis and vascular abnormalities that affect the skin and joints and progress over time to involve internal organs, most commonly the esophagus, lower gastrointestinal tract, lungs, heart, and kidneys

Mixed Connective Tissue Disease (MCTD)

Other Systemic Autoimmune Rheumatic Disease (SARDS)

overlap syndrome of limited cutaneous SSc combined with clinical features of SLE, polymyositis, and RA

Other Inflammatory myopathies

Other Systemic Autoimmune Rheumatic Disease (SARDS)

group of diseases characterized by chronic inflammation of the skeletal muscles (“myositis”) and progressive muscle weakness

Granulomatosis with Polyangiitis

• Inflammation of blood vessels

• Primarily affects the upper respiratory tract, lungs, and kidneys

Nasal or oral inflammation

chest X-ray

microhematuria

biopsy

Granulomatosis with Polyangiitis

• Laboratory Diagnosis

  • ? with oral ulcers or purulent or bloody nasal discharge

  • Abnormal ?, showing presence of nodules, fixed infiltrates, or cavities

  • Urinary sediment with ? or RBC cast

  • Granulomatous inflammation or ?

Hashimoto’s

HLA – DR4 and DR5

Organ-Specific

Autoimmune Disease	Autoantibodies
	Thyroglobulin Thyroid microsome Second colloid antigen (CA2) Thyroid membrane receptors T3 and T4

Grave’s

Organ-Specific

Autoimmune Disease	Autoantibodies
	Thyroid peroxidase antibody (TPO) TSH receptor antibody (TRAb)

IDDM

• HLA – DR3, DR4, DQ2, DQ8

Organ-Specific

Autoimmune Disease	Autoantibodies
	Insulin autoantibodies (IAA) Glutamic acid decarboxylase autoantibodies (GAD) Islet cell antigen – 2 (IA-2)

Celiac Disease

• Environmental Trigger: Gluten

• HLA-DQ2 and HLA DQ8

Organ-Specific

Autoimmune Disease	Autoantibodies
	Anti-Tissue Transglutaminase Endomysium (EmA) Deaminated Gliadin peptides

Autoimmune Hepatitis

Organ-Specific

Autoimmune Disease	Autoantibodies
	AIH-1: Smooth muscle antibodies; Anti-nuclear antibodies AIH-2: Anti-liver kidney microsomal antibody (anti-LKM-1), antiliver cytosol type 1 antibody (anti-LC-1)

Primary Biliary Cholangitis

Organ-Specific

Autoimmune Disease	Autoantibodies
	Anti-mitochondrial antibody (AMA)

Autoimmune Gastritis

Organ-Specific

Autoimmune Disease	Autoantibodies
	Antibody against parietal cells H+/K+-ATPase proton pump Cobalamin – absorbing protein

Pernicious Anemia

Organ-Specific

Autoimmune Disease	Autoantibodies
	Anti-IF or anti-parietal cells

Multiple Sclerosis

Organ-Specific

Autoimmune Disease	Autoantibodies
	Antibodies to myelin basic protein

Inflammatory Bowel Disease

Organ-Specific

Autoimmune Disease	Autoantibodies
	Deoxyribonuclease – sensitive perinuclear ANCA Anti-saccharomyces cerevisiae antibody Pancreatic antibody Anti-outer membrane porin form E.coli

Myasthenia Gravis

Organ-Specific

Autoimmune Disease	Autoantibodies
	Acetylcholine receptor p binding ab

Anti-Glomerular Basement Membrane Disease

Organ-Specific

Autoimmune Disease	Autoantibodies
	Anti-basement membrane ab

Tumor Immunology

Study of relationship between the immune system and cancer cells

Apoptosis

Tumor

Neoplasm

Benign

• ? – physiologic cell death

• ? – “to swell”

• ? – “new growth”

• ? – slowly growing cells that are well differentiated and organized, similar to the normal tissue from which they originated

Malignant

Metastasis

Cancer

Carcinoma

• ? – disorganized masses that are rarely encapsulated, allowing them to invade nearby organs and destroy their normal architecture

• ? – ability of cells to break away from the original tumor mass and spread through the blood to nearby or distant sites in the body

• ? – named after the Latin word for “crab,” derives its name from this property of invasiveness, which can resemble the legs of a crab when viewed in microscopic tissue sections

• ? – derived from the skin or epithelial linings of internal organs or glands

Sarcomas

Tumor Markers

• ? – derived from bone or soft tissues such as fat, muscles, tendons, cartilage, nerves, and blood vessels

• ? – biological substances that are found in increased amounts in the blood biological substances that are found in increased amounts in the blood, body fluids, or tissues of patients with a specific type of cancer

Alpha-fetoprotein (AFP)

Alkaline Phosphatase (ALP)

CA 15-3

CA 125

Carcinoembryonic Antigen (CEA)

Tumor Marker	Associated Cancer
	Hepatic and testicular cancer
	Lung cancer
	Breast cancer
	Ovarian cancer
	Colorectal cancer

Prostate Specific Antigen (PSA)

Nuclear Matrix Protein

Amylase

Calcitonin

CD-45

Tumor Marker	Associated Cancer
	Prostate cancer
	Urinary bladder cancer
	Pancreatic cancer
	Medullary cancer
	Hematopoietic malignancies

Acute Lymphocytic Leukemia (ALL)

Immunoproliferative Disorders

• Characterized by the presence of very poorly differentiated precursor cells (blast cells) in the bone marrow and peripheral blood

• Usually seen in children between 2 and 5 years of age and is the most common form of leukemia in this age group

CALLA (CD10)-expressing precursor B-cell ALL

Pre-B cell ALL without CALLA (CD10)

T-cell ALL

Mature B-cell ALL

Immunoproliferative Disorders

ALL

• 4 types

Chronic Lymphocytic Leukemia or Lymphoma

Immunoproliferative Disorders

• Group of diseases almost exclusively of B-cell origin

• Includes: Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL)

Hairy Cell Leukemia

Immunoproliferative Disorders

• Rare, slowly progressive disease characterized by infiltration of the bone marrow and spleen by leukemic cells without the involvement of lymph nodes

• Splenomegaly is striking, whereas lymphadenopathy is generally absent

• Malignant lymphocytes are round and often have irregular “hairy” cytoplasmic projections from their surfaces

• CD103

Hodgkin Lymphoma (HL)

Immunoproliferative Disorders

• Most common lymphomas

• Often-curable disease occurs both in young adults and the elderly

• Characterized by the presence of Hodgkin and Reed-Sternberg (RS) cells in affected lymph nodes and lymphoid organs

Non-Hodgkin Lymphoma (NHL)

Immunoproliferative Disorders

• Patients are greater than 60 years of age and the incidence is greater in men than women

• Immunosuppression: Greatest risk factor

Multiple Myeloma

Immunoproliferative Disorders

• Malignancy of mature plasma cell

• Most serious

• Bence Jones Proteins

  • Monoclonal light chains can be found in the blood, but are rapidly excreted in the urine

  • Excess of kappa or lambda light chains

Waldenström’s Macroglobulinemia

Immunoproliferative Disorders

• Malignant proliferation of IgM-producing lymphocytes

• Elevated serum monoclonal protein: macroprotein or IgM paraprotein

• In 10 to 20 percent of patients, the IgM paraproteins behave as cryoglobulins

Streptococcus pyogenes

Group A

M protein

Group A Streptococcal Infection

• Caused by ?

• Lancefield Group: ?

• ?: Major virulence factor

  • Inhibits phagocytosis

  • Diminishes complement activation

  • Attaches to host cell

Pharyngitis / tonsillitis

Impetigo / cellulitis

Group A Streptococcal Infection

• Clinical Manifestation

  • Upper respiratory tract infection: ?

  • Skin Infection: ?

  • Streptococcus Toxic Shock Syndrome

  • Otitis media

  • Puerperal sepsis

  • Sinusitis

  • Septic arthritis

  • Acute bacterial endocarditis

  • Meningitis

Acute Rheumatic Fever (ARF)

Acute Post-Streptococcal Glomerulonephritis (APSGN)

Group A Streptococcal Infection

• Damaging Sequelae

  • ? – sequela to pharyngitis or tonsillitis

  • ? – characterized by damage to the glomeruli in the kidneys

Rapid antigen detection test (RADT)\

Antistreptolysin O (ASO) titer / anti-DNase B

Group A Streptococcal Infection

• Laboratory Diagnosis

  • Culture

    • Screening Test: ?

    • Confirmatory Test: ?

  • Detection of Group A Streptococcal Antigens

    • Detection of bacterial, viral, fungal, and parasitic antigens in clinical samples

Antistreptolysin O (ASO) antibodies

Anti-DNase B antibodies

Anti-hyaluronidase antibodies

Anti-streptokinase antibodies

Group A Streptococcal Infection

• Detection of Streptococcal Antibodies

  • Diagnostically Important Antibodies

    • ? – against streptolysin O; useful for recent pharyngitis

    • ? – against deoxyribonuclease B; useful for skin infections

    • ? – less commonly used; supportive evidence of GAS infection

    • ? – supportive, sometimes measured in complicated infections

Streptozyme Testing

Group A Streptococcal Infection

• Excellent screening tool

  • Anti-streptolysin (ASO)

  • Anti-hyaluronidase(AHase)

  • Anti-streptokinase (ASKase)

  • Anti-nicotinamide-adenine dinucleotide (anti-NAD)

  • Anti-DNAse B antibodies

Anti-Streptolysin O Testing

Group A Streptococcal Infection

• Detect antibodies to the streptolysin O produced by Group A streptococcus

• Based on the ability of antibodies in the patient’s serum to neutralize streptolysin O and prevent hemolysis of red blood cells

Red cell

SLO

>240

>320

Group A Streptococcal Infection

• Anti-Streptolysin O Testing

  • Titer: Reciprocal of the highest dilution demonstrating no hemolysis

    • _________________

    • _________________

  • Controls:

    • ? control tube

    • ? control Tube

  • ASO titer: Moderately elevated if the titer is ? Todd units in an adult , ? Todd units in a child

ASO Titer Test

Todd units or IU/mL

Group A Streptococcal Infection

• Automated procedure that provides rapid, quantitative measurement of ASO titers

• 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

• Reporting: In ?

Anti-DNase B Testing

1 and 2v

Group A Streptococcal Infection

• Highly specific for group A streptococcal sequelae

• If anti-DNase B antibodies are present, they will neutralize reagent DNase B, preventing it from depolymerizing DNA. Presence of DNase is measured by its effect on a DNA methyl-green conjugate. This complex is green in its intact form, but when hydrolyzed by DNase, the methyl green is reduced and becomes colorless

• Tubes are graded for color, with a 4 indicating that the intensity of color is unchanged, and a 0 indicating a total loss of color. The result is reported as the reciprocal of the highest dilution demonstrating a color intensity of between ?

Helicobacter pylori Infection

• Agent of gastric and duodenal ulcers

• Gastric carcinoma

Spiral

Urease

Flagella

CagA

Vacuolating Cytotoxin (VacA)

Helicobacter pylori Infection

• H. pylori is able to survive and multiply in the gastric environment:

  • ? shape and flagella

  • ?

  • ? are coated with a flagellar sheath

• Virulence factor:

  • ?

    • Dysfunction of the cells signal transduction pathway

  • ?

    • Codes for a toxin precursor

Endoscopy and biopsy

Helicobacter pylori Infection

• Diagnosis:

  • Invasive Detection Methods: ? and ?

IgG, IgM, IgA

Helicobacter pylori Infection

• Diagnosis:

  • Noninvasive Detection Methods

    • Urea breath testing

    • Enzyme or lateral flow immunoassays

    • Molecular tests for H. pylori DNA

    • Serological Test: Detection of Helicobacter pylori antibodies

      • Primary Screening Method

        • (3)

      • Antibody testing is not as well suited for determining eradication of infection as are other methods

Mycoplasma pneumoniae Infection

• Class Mollicutes

• Leading cause of upper respiratory infections worldwide

I antigen

4 °C

37 °C

Mycoplasma pneumoniae Infection

• Cold isoagglutinins

  • IgM antibodies directed against the ? found on the surface of human RBCs

  • Agglutinate the RBCs at ?

  • Reaction is reversible when the samples are warmed to ?

  • IM, CMV Infection

Trypticase soy broth

Mycoplasma pneumoniae Infection

• Laboratory Diagnosis of Mycoplasma pneumoniae infection

  • Culture: ? with 0.5% albumin, SP4 medium, or a viral transport medium

  • Delay: Slow growth—colonies may take 1–3 weeks to appear

PCR (Polymerase Chain Reaction)

Mycoplasma pneumoniae Infection

• Molecular Diagnosis: ? for the diagnosis of Mycoplasma infections

Rickettsia prowazekii

Rickettsia typhi

Orientia tsutsugamushi

Diseases	Rickettsial Agent	Insect Vectors
Typhus Group Epidemic Typhus Murine Typhus Scrub Typhus	?	Louse Flea Mite

Rickettsia conorii

Rickettsia rickettsii

Rickettsia akari

Diseases	Rickettsial Agent	Insect Vectors
Spotted Fever Group Indian Tick Typhus Rocky Mountain Spotted Fever Rickettsial Pox	?	Tick Tick Mite

Coxiella burnetii

Bartonella quintana

Diseases	Rickettsial Agent	Insect Vectors
Others Q Fever Trench Fever	?	Nil Louse