immunofluorescence and autoimmunity

lower, longer

energy emitted from photon is higher/lower energy and shorter/longer wavelength than absorbed

fluorescein isothiocyanate (FITC)

most common fluorochrome

-green

direct fluorescence (DIFA)

-detects Ag

-patient tissue w Ag + fluorescently labeled Ab → (incubate and wash) → fluorescence if Ag present

Ag

direct fluorescence tests for Ag/Ab

indirect fluorescense (IFA)

-detects Ab presence

-manufactured Ag + patient serum w Ab → (inc/wash) → conjugate (f labeled Ab to human Ig) → (inc/wash) → fluorescence directly proportional to amt patient Ab in sample

Ab

indirect fluorescence tests for Ag/Ab

autoantibodies

antibodies produced by immune syst that mistakenly target and react w person’s own tissues and organs

-circ immune complexes cause autoimmune disease

-organ specific, person specific, systemic

substrates

-used in lab to detect auto-Ab

-animal/human tissue, organs, cells

-rat kidney, stomach, liver

-microorg: Treponema, Mycoplasma, Legionella

conjugate

-fluorescent labeled Ab

-typically FITC

anti-nuclear Ab (ANA)

-titer

-uses Hep2 cells

-read for fluorescence

-patterns are found with resting and mitotic states

-homogeneous, speckled, nucleolar, centromere

high

ANA titer high/low with:

-RA

-SLE

-MCTD

-scleroderma

-dermatomyositis

-malignancies

-hypersensitivity

-Sjogren’s synd

low

titer high/low with:

-aging

-viral diseases

-chronic hepatitis

-some people just have

greater

titers greater/less than 80 are considered significant

homogeneous

ANA pattern

-resting: total fluorescence

-mitotic: green cigar

-dsDNA, chromatin

total, green cigar

resting and mitotic descriptions for ANA homogenous pattern

dsDNA, chromatin

homogenous pattern seen with Ab to …

speckled

ANA pattern

-resting: dark dots

-mitotic: brown cigar

-SSa/SSb, Scl-70, Sm, RNP, ribosomal p, Jo-1, ANoAs

dark dots, brown cigar

resting and mitotic descriptions for ANA speckled pattern

SSa/SSb, Scl-70, Sm, RNP (ribosomal p, Jo-1, ANoAs)

speckled ANA pattern seen with Ab to…

nucleolar

ANA pattern

-resting: green blobs

-mitotic: green cigar

-nucleolus

green blobs, green cigar

resting and mitotic descriptions for ANA nucleolar pattern

nucleolus

nucleolar ANA pattern seen with Ab to …

centromere

ANA pattern

-resting: green dots

-mitotic: patchy center

-centromere B

green dots, patchy center

resting and mitotic descriptions for ANA centromere pattern

centromere B

centromere ANA pattern seen with Ab to…

BioPlex 2200

-looking for Ab

-magnetic beads infused with fluorescent dye and coated w Ag

-flow cytometer

-if Ab pos, assume ANA pos

Ab

looking for Ag/Ab with BioPlex 2200

anti-DNA

Ab 

-uses Crithidia luciliae as substrate

-kinetoplast fluoresces when pos for Ab

kinetoplast (dsDNA)

fluorescent in pos test for anti-DNA test

Crithidia luciliae

substrate for anti-DNA testing

anti-mitochondrial Ab (AMA)

-positive test in primary biliary cirrhosis

-substrate: rat kidney

-”loopy”

rat kidney

AMA substrate

primary biliary cirrhosis

condition with positive AMA

anti-smooth muscle Ab (ASMA)

-pos test in chronic active hepatitis

-substrate: rat stomach

-”striated”

rat stomach

ASMA substrate

chronic active hepatitis

condition with pos ASMA 

SLE

ANA profile

-DNA: homo

-Sm: speckled

-chromatin: homo

-RNP: speckled

-SSa: speckled

-nucleolus: nulceolar

SLE

-systemic

-unknown cause (hormonal, genetic, eniron, drugs) → mostly women

-low grade fever, fatigue, anorexia, musc/joint pain

-butterfly rash

-renal (anti-DNA Ab assoc), CNS, caridac, resp involvement

-Raynaud’s phenom

-dec complement (C4, CH50)

-95% ANA

-DNA, Sm, chromatin (RNP, SSa, nucleolus)

Sjogren’s syndrome

-seen with other diseases (RA)

-systemic

-sicca complex: eye/mouth dryness, arthritis

-auto-Ab to salivary/tear glands

-women >40

-ANA: SSa/SSb (nucleolus)

Sjogren’s syndrome

ANA profile

-SSa/SSb: speckled

-nucleolus: nucleolar

mixed connective tissue disease (MCTD)

-SLE symptoms

-hand swelling

-women

-rarely renal involvement

-ANA: RNP 

MCTD

ANA profile

-RNP: speckled

scleroderma

-progressive systemic sclerosis

-hardening and tightening of skin and connective tissue

-diffuse: severe

-CREST: milder, Raynaud’s

diffuse

-scleroderma type

-severe

-ANA: Scl-70 (speckled); nucleolus (nucleolar)

CREST

-scleroderma type

-milder

-Raynaud’s common

-ANA: centromere

Goodpasture’s

-glomerular basement membrane Ab (GBM)

-respiratory and kidney involvement

-glomerulonephritis and pulmonary hemorrhage

glomerular basement membrane (GBM)

auto-Ab in Goodpasture’s

pernicious anemia

-gastric parietal cell Ab

-anti-IF Ab → B12 def → megaloblastic anemia

parietal cell (IF)

auto-Ab in pernicious anemia

drug induced LE

-auto-Ab to histone

-isoniazid, nitrofurantoin, procainamide, hydralazine

-goes away after use

-ANA: homogenous

-no renal or CNS involvement

-rat stomach: pos

homogeneous

ANA profile for drug-induced LE

histone

auto-Ab in drug induced LE

rat stomach

substrate positive in pernicious anemia

type I diabetes

-kids

-insulin dependent

-HLA assoc: DR3, DR4

-auto Ab to beta cells of islets of Langerhans

beta cells

auto-Ab in type I diabetes

anti neutrophil cytoplasmic Ab (ANCA)

-auto-Ab to PMN granules

-autoimmune vasculitis assoc (granulomatosis polyangitis - Wegner’s granulomatosis; microsscopic polyangitis)

-C-ANCA: cytoplasmic staining pattern

-P-ANCA: perinuclear staining pattern

nuclear border

fluoresces in P-ANCA

cytoplasm

fluoresces in C-ANCA