Immunofluorescence & Autoimmunity

Most common fluorochrome

FITC

Direct Fluorescence Steps (7)

light source

heat filter

exciter filter

condenser

slide

barrier filter

eye

Immunofluorescence Assay

1. tissue with Ag

2. patient serum auto-Ab

3. binds with fluorescent Ab to human globulin

4. fluoresce

Autoantibody substrates (3)

anima/human tissue, organs, or cells

rat kidney, stomach, or liver

microorganisms (Treponema, Mycoplasma, Legionella)

Anti-nuclear Antibody (ANA)

elevated/positive in SLE< RA< MCTD< scleroderma, dermatomyositis, malignancies, and hypersensitivity (autoimmune disorders)

low titers seen in elderly, viral diseases, chronic hepatitis

ANA developed against (6)

dsDNA

SSa (Sjogrens syndrome A)

SSb (Sjogrens syndrome B)

Sm (Smith)

RNP

nucleolar RNA

ANA testing procedure

looking for pt. Ab

• Hep-2 cells fixed on slides

• add serum + incubate

• wash, add conjugate, incubate

• wash + counterstain

• read for fluorescence

ANA Patterns (4)

homogeneous (anti-histone)

speckled (anti-Sm)

nucleolar (anti-RNA)

centromere (anti-centromere)

ANA Confirmatory Testing (5)

SSa/b

Scl-70

Sm

RNP

ds DNA

Additional ANA Confirmatory Testing (5)

chromatin (homo)

centromere B (centromere)

ribosomal P

Jo 1 (speckled)

ANoAs

BioPlex 2200 does or does not screen directly for ANA?

does NOT

Anti-DNA Test substrate

Crithidia luciliae

Parts of Crithidia luciliae examined for (2)

kinetoplast (rich in DNA)

nucleus

Crithidia luciliae is a _________flagellate

hemo

Anti-Mitochondrial Antibody

• rat ___________ substrate

• primary biliary _____________

kidney; cholangities

Anti-Smooth Muscle Antibody (ASMA)

• rat ____________ substrate

• _______________ active hepatitis

• primary biliary _________________

stomach; chronic; cholangitis

Anti-Parietal Cell Antibody (APCA)

• rat ______________ substrate

• _________________ anemia

stomach; pernicious

Anti-neutrophil cytoplasmic antibodies (ANCA)

• antibodies to ___________ in PMNs

• associated with:

  • autoimmune _______________

  • ______________ granulomatosis

  • microscopic ________________

• P-ANCA, C-ANCA

granules

vasculitis

Wegener’s

polyangitis

Systemic Lupus Erythematosus (SLE) (2)

affects mostly women

systemic disease

SLE Causes (4)

mainly unknown, but they look at:

• hormonal

• genetic

• environmental

• drug-induced

SLE symptoms (3)

Reynaud’s phenomenon

butterfly rash (face)

fever

(+more)

SLE Test Results (4)

decreased C4

decreased CH50

positive DNA antibody

positive ANA

SLE ANA profile (5)

ANA

DNA

ENA

Scl-70

SSa/SSb

Decrease in complement can lead to ______

SLE

Sjogren’s Syndrome (5)

seen in conjunction with other diseases (RA)

women

systemic

Sicca complex (dry eyes + mouth, due to Ab attack on salivary and tear glands)

SSa+, SSb+

Mixed Connective Tissue Disease (MCTD) (3)

sim. symptoms to SLE (swelling in hands)

women

RNP+

Scleroderma

fibrosis + degenerative change in skin, synovium, internal organs (progressive systemic sclerosis)

diffuse

CREST: milder form

CREST Scleroderma

Calcinosis

Reynaud’s

Esophageal

Sclerodactyly

Telangiectasises

Goodpasture’s (3)

Ab to glomerular basement membrane

2/3 have GBM+ antibodies

glomerulonephritis + pulmonary hemorrhage

Drug Induced LE (3)

drugs: isoniazid, nitrofurantoin, procainamide, hydralazine

homogenous ANA

no renal/CNS involvement

Type 1 Diabetes

assoc. with HLA-DR3 & DR4

Antibody: DNA

Pattern: __________________

Disease: ______________

homogeneous

SLE

Antibody: RNP

Pattern: ____________

Disease: ______________

speckled

MCTD/SLE

Antibody: Sm

Pattern: ___________________

Disease: ____________________

speckled

SLE

Antibody: Scl-70

Pattern: ___________________

Disease: ____________________

speckled

scleroderma

Antibody: SSa

Pattern: ___________________

Disease: ____________________

speckled

Sjogren’s A

Antibody: SSb

Pattern: ___________________

Disease: ____________________

speckled

Sjogren’s B

Antibody: Centromere

Pattern: ___________________

Disease: ____________________

centromere

CREST variation of scleroderma

Antibody: Nucleolus

Pattern: ___________________

Disease: ____________________

nuclear

scleroderma, Sjogren’s, SLE

Antibody: Chromatin

Pattern: ___________________

Disease: ____________________

homogeneous

Lupus