Immnofluorescence & Autoimmunity

Direct fluorescence

Tagged labeled antibody is added to patient tissue antigen/sera to detect antibody

Indirect fluorescence

detects specific antibodies in a patient's serum by using a fluorescently labeled secondary antibody to visualize the binding of the primary antibodies (from the patient's sample) to a known antigen

Use of a conjugate

Autoantibody testing

Antibodies against self cell, circulating immune complex cause auto immune disease

Substrates = animal or human tissue, organs, or cells

Rat kidney/stomach/liver

Treponema, Mycoplasma, Legionella

FITC

an antibody that has been conjugated (labeled) with the fluorescent molecule fluorescein isothiocyanate (FITC), or an antibody that recognizes FITC itself

To visualize and quantify specific molecules or cells

Flow cytometry

ANA antinuclear antibody

Develop into antibody to

DsDNA

SSa

SSb

Sm

RNP

Nucleolar RNA

ANA elevated in

SLE, RA, MCTD, scleroderma, dermatomyositis, malignancies and hypersensitivity

ANA low titers in

Aging, certain viral diseases, chronic hepatitis

Dilute sample to reduce errors

ANA procedure

Hep-2 cells are fixed on slides

Serum added and incubated

Washed then conjugate added

Washed and counterstained = Specific patterns

dsDNA pattern

Homogenous pattern = SLE

RNP pattern

Speckled = MCTD/SLE

Sm (smith)

Speckled = SLE

Scl-70 pattern

Speckled = scleroderma

SSa pattern

Speckled = SLE/ Sjogren’s

SSb pattern

Speckled = Sjogrens

Centromere pattern

Centromere = CREST variant, Scleroderma

Nucleolus pattern

Nucleolar = Scleroderma, Sjogrens or SLE

Chromatin pattern

Homogenous = SLE

Crithidia luciliae

Substrate, Hemoflagellate

Kinetoplast rich in DNA = identify dsDNA antibody

Brighter fluoresence

AMA

Primary biliary cirrhosis

Antibodies against kidney

ASMA

Chronic active hepatitis

Antibodies against rat stomach/mucosa/ smooth muscle

SLE Systemic Lupus Erythematosus

Systemic disease, mainly in fenales

Butterfly rash, renal, CNS, cardiac/respiratory

Reynauds phenomenon

SLE causes

Multiple ANA patterns

Hormonal, genetic, environmental, drug induced

SLE test results

Decreased C4, CH50

Positive DNA antibody

ANA, DNA, ENA (Sm/RNP), Scl-70, SSa/SSb

Sjogrens syndrome

May be seen in conjunction with Rheumatoid arthritis

Systemic

Sicca complex, more common in women

Dry eyes/mouth, arthritis

Antibodies to salivary/tear glands = SSa/SSb

MCTD Mixed connective tissue disease

Symptoms similar to SLE, swelling in hands

Most common in women

95% = +ve RNP

Scleroderma

Fibrotic and degenerative changes in skin, synovium and internal organs

Hardening and tightening of skin and connective tissue

Scl-70, Nucleolar pattern ANA, multiple body systems

CREST variant = skin, hand, face

CREST variant scleroderma

Centromere pattern

Calcinosis = calcium deposits

Raynaud’s phenomenon = spasm of blood vessels

Esophageal dysfunction = acid reflux

Sclerodactyly = thickening and tightening of skin

Telangiectasis = Dilation of capillaries, red marks

Goodpastures syndrome

AMA = Rat kidney

Glomerular basement membrane antibodies

Basement membrane antibodies in respiratory tract

Glomerulonephritis and pulmonary hemorrhage

Pernicious anemia

Gastric parietal cell antibody

Intrinsic factor B12 = Needed for DNA production = Megaloblastic anemia

ASMA test = Smooth muscle/stomach

Drug induced SLE

Isoniazid, nitrofurantoin, procainamide, hydralazine

Homogenous ANA

No renal or CNS involvement

Type 1 diabetes

Insulin dependent diabetes

HLA DR3 and DR4, P-ANCA

Destruction of insulin producing beta cells of islets of langerhans

Autoantibodies to beta cell antigens

ANCA anti neutrophil cytoplasmic antibodies

Antibodies to granules in PMNs

Associated with autoimmune vasculitis

Wegener’s granulomatosis

C-ANCA

P-ANCA

C-ANCA

Cytoplasmic staining pattern

P-ANCA

Perinuclear staining pattern