Hypersensitivity and Precipitation / Agglutination

Hypersensitivity

Adverse effects of immune system that can result in ilness

Type 1 hypersensitivity

IgE mediated hypersensitivity or immediate

Anaphylaxis - against protection

Local - hay fever or hives

IgE stimulates basophils and mast cells = degranulate

Systemic - contractions, decreased BP, organ failure

Type 1 examples

Insect stings - heymenopterae - ants, bees, wasp

Haptens - RXN when bound to protein

Type 1 treatment

Vasopressor

Bronchodilator - theophylline

Epi pens

Type 1 mediator release

Activation of platelets, neutrophils, and eosinophils release histamine

Type 1 atopy

hay fever or asthma; hypersensitivity reaction with genetic predisposition

Type 1 ImmunoCap

Measures total IgE against a specific allergen, skin test

Type II hypersensitivity

IgG or IgM mediated or cytotoxic response

Ig reacts with cell-associated antigen, causing complement to be activated

Immediate transfusion RXN

HDFN

Treatment of some drugs

AIHA (Autoimmune hemolytic anemia)

Type III hypersensitivty

Immune complex formation

Soluble antigen + soluble antibody (primarily IgG) form a precipitate

Complexes formed activating complement and causing inflammation

Immune complex glomerulonephritis, SLE, Rheumatoid arthritis

Type IV hypersensitivity

T cell mediated, Delayed T cell hypersensitivity, 48 - 72 hours after exposure

No antibodies involved

Prev exposed T cells come in contact with antigen

Type IV examples

Release of cytokines

Contact dermatitis = poison ivy

Tuberculin reaction = Patch test, skin test

Precipitation

Soluble antigen + soluble antibody joins to form an insoluble precipitate

Ouchterlony

Radial immunodiffusion

Aspergillus

Ouchterlony

Double diffusion of Antigen and Antibody, nothing in gel

Identity

Partial identity

Non-identity

Identity

Two antigens are immunologically identical

Partial identity

Determinants present in one antigen but lacking in the other, one “spur”

Non-identity

None of the antibodies in the serum react with antigenic determinants that may be present in both the antigens,

Two antigens are immunologically unrelated as far as that antiserum is concerned

Radial immunodiffusion

Quantitative, single diffusion

Ring is proportional to the concentration of the antigens

ANTIGEN in the well diffusing outward

GEL contains antibody conc.

Agglutination

Particulate antigen combining with antibody to form a particle clump

Particulate cary antigens 100-1000 times larger than antibody

IgM are best agglutinates, very sensitive

Precipitation

Soluble antigens + soluble antibodies = insoluble

IgG are best precipitators

More specific than agglutination

Direct agglutination

Cold agglutinins, Mycoplasma pneumoniae

Antigen ON the RBCs, at 0-20C (I blood group)

Single significant titer of 1:32 or fourfold rise in titer

Must keep warm

Indirect or Passive agglutination

Carrier for antigen on latex or manufactured RBCs, looking for antibodies

TPO antibodies

Rheumatoid factor = Usually IgM Ab to Fc portion of human IgG

CCP more useful than RF

Tanned RBCs

Hemagluttination

Indirect agglutination

Absorb antigens

Hashimotos

Indirect agglutination

TPO elevated with 90%

Thyroglobulin elevated with nearly all

Hypothyroidism,

stimulation of B cells—> plasma cells produce antibodies to destroy thymocytes + activate CD8 T cells

Graves

TPO elevated with 75%

Thyroglobulin elevated in 70%

Antibodies attach toTSH receptors = unlimited production = hyperthyroidism

Rheumatoid factor

Latex, Antibody against FC portion of IgG

Frequent in women 30-50 y/o

Latex particles are coated with human IgG antigen, 1:6 dilution

Rheumatoid factor increased in

Elderly, sarcoidosis, SLE, Sjogren’s syndrome, hepatitis, cancer, tuberculosis, syphilis

Rheumatoid factor confirmatory test

CCP (cyclic citrullinated protein) more specific for diagnosis of RA

Reverse passive agglutination

FC portion of antibody attached to carrier - latex

Detects soluble antigen

Cryptococcus neoformans

Looking for antigen

Agglutination inhibition

Inert hapten coated particles + specific antisera

Patient sera added to antisera then hapten added

Looking for antigen, presence of antigen prevents clumping

No agglutination = +ve

HIA Hemagglutination inhibition assay

Patient serum is mixed with a standardized amount of antigen.

The titer of the patient’s serum is the highest dilution that blocks agglutination

Syphilis

Treponema pallidum

Primary chancre - 1-5 weeks

Secondary - 6-8 weeks after chancre, swollen lymphs

Tertiary - 10-30 years = cardiac, CNS

Syphilis microscope

Darkfield microscope, spirochetes

Non treponemal testing

Looking for antibody to reagin

Byproduct of cell damaged and breakdown of lipids (cardiolipin) = body produces antibody (reagin)

VDRL = beef cardiolipin, cholesterol, and lecithin

Heat inactivated serum or CSF = destroy complement

Looking for flocculation

RPR Rapid plasma reagin

Non-treponemal testing

Cardiolipin, chlone chloride, EDTA, and charcoal

Swirl and look for tail

Plasma or serum no heat inactivation

CANNOT USE CSF

RPR false positive

Other treponemal infections, SLE, RA, acute viral disease, immunizations, pregnancy, old age

Treponemal testing

Detect direct antibodies to Treponema pallidum

FTA - ABS

TPPA

FTA-ABS Fluorescent treponemal antibody absorption

Reiters strain removes non specific Ab (non pathogenic)

Nicholas strain - pathogenic, specific antibodies to syphillus

Antibody-Antigen complex = glow

TPPA treponema pallidum particle agglutination

Antigen attached to stained gelatin particles

Reactive = +ve in TP well and negative in control = +ve antibody