Quiz 2 - Autoimmunity & Serological Techniques Flashcards
When autoimmunity is suspected, we perform this test…
Anti-nuclear antibodies (ANA)
Are men or women more predisposed to autoimmune disorders?
Women
When antigen-antibody complexes deposit in tissue, they attract…
Complement
- rupture/lyse cells
- autoimmune diseases cause tissue injuries
3 steps of process of disease
- Autoantibodies complex with patient’s own DNA
- Immune complexes deposited in vascular systems - organs & tissues
- Immune-mediated tissue injury due to complement
ANA vs Crithidia luciliae - which is the screening test and which is the confirmatory test?
ANA = screening
Crithidia luciliae = confirmatory
ANA test uses these cells as the substrate
HEP-2
- epithelial cell line
- ANA reacts with nuclear DNA/RNA
Why do different ANA patterns appear?
Due to different stages of nuclear DNA mitosis
How is the ANA test performed?
Patient sera placed on slide with HEP-2 cells and labeled with conjugate antibody with fluorescence tag
- ie FITC
- “sandwich” assay
The ANA test is an example of a “sandwich” assay. What is being sandwiched?
Patient’s antinuclear antibodies
-between HEP-2 DNA and conjugate antibody with fluorescence tag
An ANA test result showing a homogeneous pattern indicates this autoimmune disease
SLE = Systemic lupus erythematosus
An ANA test result showing a centromere pattern indicates this autoimmune disease
CREST syndrome
-form of scleroderma
An ANA test result showing a speckled pattern indicates this autoimmune disease
Sjogren syndrome
An ANA test result showing a nucleolar pattern indicates this autoimmune disease
Scleroderma
SLE - shows this ANA test pattern. What do the autoantibodies target (3)?
Homogeneous
anti-dsDNA
anti-ssDNA
Histones
-includes anti-Sm abs
Sjogren - shows this ANA test pattern. What do the autoantibodies target?
Speckled
anti-RNP (ribonuclear protein)
Scleroderma - shows this ANA test pattern. What do the autoantibodies target?
Nucleolar
anti-nucleolar
CREST - shows this ANA test pattern. What do the autoantibodies target?
Centromere
anti-centromere
After performing an ANA test for SLE, we confirm our results with this test. What does the confirmatory test target in the organism?
Crithidia luciliae
Kinetoplast
- mitochondria rich in dsDNA
- highly sensitive to HEP-2 substrate
The hereditary component sometimes involved in autoimmune disease involves the…
HLA
SLE - laboratory findings
Anemia - 50% leukopenia
Thrombocytopenia - 25-50%
Elevated erythrocyte sedimentation rate (ESR)
ANA positive - anti-dsDNA and anti-Sm. Targets nucleoprotein antigens
Overuse of this causes a SLE-like syndrome. The autoantibodies developed target…
Drugs
Histones
-milder SLE
SLE vs drug-induced SLE - what would the Crithidia luciliae test results be?
SLE = positive
Drug-induced SLE = negative
-absence of anti-dsDNA antibodies
SLE - clinical manifestations
Arthritis - chronic inflammation - skin, joints, kidneys, lungs, CNS
Butterfly rash across nose/upper cheeks
Raynaud’s syndrome
Renal disorders
Pulmonary manifestations
CNS disorders - depression, psychosis, convulsions
What is Raynaud’s syndrome. Which autoimmune disorders is it associated with?
Cyanosis on cold temperatures. Stress-induced vasoconstriction on extremities
SLE, CREST (the “R”)
Sjogren’s syndrome - 90% of autoantibodies are called…
Rheumatoid factor
-anti-RNP associated with ANA stain
Sjogren’s syndrome - laboratory findings
ANA - anti-La
Labial salivary gland biopsy for definite diagnosis
Dry mouth and dry eyes are symptoms associated with…
Sjogren’s syndrome
-affects lacrimal, salivary & excretory glands
Scleroderma has 2 forms
Progressive diffuse
Systemic = CREST
What are the acronyms of CREST?
Calcinosis - bone formation Raynaud - vasoconstriction of hands/feet Esophageal involvement Sclerodactyly - harden finger skin Telangiectasia - spider veins
Scleroderma - lab findings
ANA - centromere pattern (CREST) or nucleolar pattern
Insulin-dependent diabetes mellitus - insulin production is deficient due to…
Destruction of cells of pancreas
- congential rubella
- HLA
This autoimmune disease has symptoms that overlap to SLE. How are lab results different from SLE?
Mixed connective tissue disease
Absence of multiple anti-SM and anti-dsDNA
Ankylosing spondylitis - males or females more prone? What is a common symptom?
Males
Fibrosis of bone synovial capsule
Rheumatoid arthritis - primarily affects…
Synovial joints
-swelling, morning stiffness, weight loss, fatigue
Rheumatoid arthritis - lab findings
RF
-latex agglutination detects IgM RF
Circulating immune complexes
ANA - 14-28% of patients
What antibodies are expected for a hepatitis infection?
Anti-smooth muscle
What antibodies are expected for biliary cirrhosis (PBC)?
Anti-mitochondrial
What antibodies are expected for Goodpasture’s?
Anti-glomerular basement membrane
-kidney’s glomerulus damaged
What antibodies are expected for pernicious anemia?
Anti-parietal cells
IgG subclasses - which ones are autoimmune and which ones are infectious antibodies?
IgG 1 & 3 = infectious
IgG 2 &4 = autoimmune
Multiple sclerosis and myasthenia gravis are similar in that they both affect the…
Nervous system
- MS - brain, spinal cord, myelin shealth
- MG - neuromuscular
- do an MRI scan
Hashimoto’s thyroiditis vs Graves disease - both have what in common? How are they different
Enlarged thyroid
Hashimoto’s - hypothyroidism
-weight gain, bradycardia, slow metabolism
Graves - hyperthyroidism
-weight loss, tachycardia, fast metabolism
Pernicious anemia - this is prevented from being absorbed… due to the lack of this…
Vitamin B12
Intrinsic factor
Difference between infectious liver disease and non-infectious liver disease?
Non-infectious (autoimmune) = ANA towards smooth muscle proteins
Infectious = no ANA
-see anti-viral, anti-bacterial antibodies
Autoimmune liver disease - lab findings
anti-liver antibodies:
anti-liver soluble protein (anti-LSP)
anti-liver membrane antibody (anti-LMA)
anti-acidoglycoprotein receptor (anti-ASCP)
No anti-dsDNA
The highest peak on a normal SPE graph corresponds to…
Albumin
Multiple myeloma patients show a large spike in the SPE corresponding to…
gamma-globulins
What does IFE stand for? What is the test used to determine?
Immunofixation Electrophoresis
Determines what immunoglobulin abnormalities exists
After band separation, IFE uses ___ to probe for immunoglobulins and visualize bands
Anti-antibodies
Complement can be inactivated by ___
Heat
-56C for 30 mins
The complement system is activated by…
Antibodies
Name two assays to detect complement
ELISA
CH50
The CH50 assay is used to detect hemolytic activity of… What does the CH50 value mean?
Complement
Amount of patient serum required to lyse 50% of a standard concentration of sheep RBC
How is titer calculated from a dilution?
Titer is the reciprocal of the dilution
Strep toxin can cause this substrate, ___, to change from blue to pink. What does strep toxin do? If patient has antibodies against strep toxin, what color will the test be?
Toluidine blue
Depolymerize DNA = pink color
Blue
- toxin neutralized
- no color change
How is the dilution calculated from the dilution factor?
Reciprocal
-dilution factor 5 is 1:5 dilution or 1/5
Antigen-antibodies can form 3 zones depending on their concentration. What are the 3 zones as it relates to the amounts of antibodies and antigen?
Prozone = too much antibody
Zone of equivalence
Postzone = too much antigen
Precipitation test uses the concept of… using this media…
Immunodiffusion
Agar plate
3 types of agglutination tests
Latex agglutination - mono, RA, rubella
Hemagglutination
Inhibition reactions
Difference between turbidimetric and nephelometric
Turbidimetric = measure light absorbance by immune complex
Nephelometry = measure light scatter by immune complex
Immunodiffusion assays have this weakness. However, they’re still useful for detecting ___ infections
Takes a long time to do
Fungal
A double immunodiffusion assay is also called… What are the patterns that form?
Ouchterlony
Identity
Partial Identity
Non-identity
Describe what is happening in each Ouchterlony assay reaction
Identity - both antigen/antibody and antigen/unknown have matching epitopes and will diffuse radially towards one another
Partial identity - share a few epitopes, but have more not in common. Forms small bifurcation/spur
Non-identity - precipitation lines cross, do not share epitopes
Radial Immunodiffusion (RID) - describe assay. What does the result look like?
Antibody infused into agar. Inoculate patient sample into well and let diffuse out to zone of equivalence
Zone of equivalence = precipitation ring
-ring diameter proportional to concentration
IEP vs IFE - which is more sensitive and produce sharper bands? How are they similar?
IFE
Both use electrophoretic immunodiffusion
Terms for when Igs in a patient are lower than normal, higher than normal, and none
Hypogammaglobinemia
Hypergammaglobulinemia
Agammaglobulinemia
Agglutination tests that use RBCs in the assay are called…
Hemagglutination tests
Agglutination assays can be direct or indirect. What does each one mean?
Direct - antigen-antibody bind directly
Indirect - antigens are coated on a carrier molecule
-makes reaction more visible, easier to interpret
Flocculation assay is a subset of this type of test… It is rarely used besides detection of this disease…
Agglutination
-precipitation of fine particles
Syphilis
-VDRL and RPR
Describe the complement fixation test. Describe the positive and negative results
Complement is “fixed” with antigen-antibody so it cannot react and lyse RBCs
-uses sheep RBCs
Positive = antigen-antibody complex present, no lysis Negative = antigen-antibody complex not present, lysis
Immunoblot - difference between southern blot and western blot
Southern = DNA
Western = protein
Define heterophile antibodies. An example of a heterophile antibody is known as…
Antibodies that react with antigens not responsible for their production = non-specific
Forssman antibody
- IgM in nature
- made against guinea pig kidney but reacts with sheep RBCs
2 different tests to detect heterophile antibodies
Paul Bunnell heterophile antibody test
Davidsohn differential test
