Immunofluorescence Flashcards
What are the principles of fluorescence when applied to microscopy?
When the concept of fluorescence is applied to microscopy it can involve labelling antibodies with fluorescent molecules in order to visualise the antibodies down the microscope.
Most commonly used fluorochrome in routine clinical immunology lab is Fluorescein Isothiocyanate (FITC).
The basic principles are as follows:
Submit specimen to specific band of wavelengths - collect emitted light and observe specific pattern
Separate emitted light from excitation wavelength using a dichromatic mirror
Excitation filter and emission filters also required, selective for wavelengths
What are the uses of HEp2 cells in immunofluorescence?
Hep2 cells (derived from laryngeal cancinoma (human source)) are the commonly used substrate for immunofluorescence, previously used rodent liver:
- Human origin – more specific for the antibody
- Improved sensitivity from antigen expression
- Larger nuclei, see clear patterns
- Monolayer of cells
- High rates of cell division
- Non-specific substrate improves sensitivity (better screening)
What is fluorescence?
The property of some molecules to absorb light at one wavelength and emit at a longer wavelength after excitation.
This change in wavelength is referrred to as ‘The Stoke’s Shift’ (1852. Sir George Stokes).
Molecule absorbs photons from UV/Vis light spectrum causing transition to a high electron state. Emit photons when it returns to the original state.
How does the equipment used permit the principles of fluorescence?
Light source - LED is safer than old mercury, gives off various wavelengths
Excitation filter only lets through the specific wavelength we’re interested in for it to excite the fluorochrome.
A mirror reflects this through the objective onto the specimen.
The specimen, if it contains antigens or antibodies of interest, will fluoresce letting off a different wavelength of light. This passes through the mirror and the ocular into the detector.
In the case of microscopy this detector is usually the person reading the results. Hence what you can visualise down the microscope.
What are the problems with fluorescence?
Choice of fluorophore (usually FUTC)- Excitation/emission difference
Concentration of fluorophore - dilution used to optimise test (best pic at lowest dilution (lowest cost))
Magnification (not usually an issue in clinical labs. Increase has a quenching effect)
Bleaching / degradation of fluorophore over few days
Background / autofluorescence - can get non-specific patterns down microscope
What are the different uses of fluorescence?
Visualise individual proteins eg. Components of endothelial cells.
Molecular associations on living cells – protein to protein interactions, fluoresce different proteins in different colours
In-vivo trafficking – eg. cancer cell movement in blood vessels
Study cellular activities such as channel opening / closing, ion movements
Genetic information in samples (target genomic sequences labelled) eg. FISH - using specific probes for specific parts of the chromosome allows precise resolution of morphological and genomic structures
What is the process of indirect immunofluorescence?
- Allow reagents to equilibrate to room temperature - as per manufacturers instructions - antibodies don’t work well in the cold so wont bind optimally.
- Organise samples to be tested and create the worksheet -which patient sample is going in which well (can be automated). Then dilute appropriately.
- Prepare slides (must be at RT) – label (i.e. date and 1, 2, 3…) or link barcode to worksheet - can be automated, so all information is held in a system and can be recalled.
- Add controls and samples - each well has some cells in dd diluted patient sample/control to it. Avoid spilling using a hydrophobic lining on cell, this places a droplet on only that cell - avoids cross-contamination.
- Place in humidity chamber & incubate - usually for 30 min - avoids sample drying out.
- Wash - one by one to avoid cross contamination.
- Add conjugate (fluorochrome labelled antibody) and incubate - 30 min to allow antibodies to bind.
- Place in humidity chamber & incubate.
- Wash- remove excess.
- Mount - maintain fluorescence intensity.
- Visualise down a microscope, or use automation to visualise for you.
This is indirect, where the tissue specimen is on the slide eg adrenal gland tissue, hep2 cell line, etc. Then add patient sample, if this contains antibodies to any target antigens on the tissue (those you’re interested in) it will bind.
What is direct immunofluorescence?
The tissue sample is from the patient, eg skin biopsy or renal biopsy. Tissue is added to the slide and then the fluorochrome-cells conjugated antibody is added directly.
Then wash off any excess that has not bound and observe the fluorescence.
If there is complementary antigen in sample, the labelled antibody will bind and fluoresce - this is what you will see down microscope.
What are autoantibodies?
Antibodies inappropriately directed against ‘self’
Results in inflammation
Genetic predisposition – HLA type- eg HLA B27 leads to rheumatoid diseases.
Environment – chemicals, infections, diet
System or organ targeted varies
- Single organ disorders - eg autoimmune thyroid disease
- Systemic disorders - eg fatigue, joint issues…
Polyclonal
Vary in specificity to disease – cross reactivity
Vary in isotype – G, A, M
Vary in affinity and avidity (May have a positive test result, but binding is weak so there are no symptoms)
- Affinity = strength of individual interaction
- Avidity = overall strength of complex (multiple binding, and the conformation of the epitope)
Tests are highly variable - there is a lack of standardisation between tests, so must piece together with clinical picture
What are antinuclear antibodies?
Most commonly requested test
IgG autoantibodies against nuclear or cytoplasmic components of cells
Positive result followed up with testing for antibodies to specific antigens (Extractable Nuclear Antigens or DNA)
Clinical specificity varies highly
Not always associated with autoimmune disease - disease associated not disease causing
Some individuals may be seronegative - negative result doesn’t mean they don’t have it.
Associated with many diseases Systemic: SLE MCTD - mixed connective tissue disease RA Sjogrens syndrome…
Other disorders: Autoimmune liver disease Chronic infection Vasculitis Elderly…
Which disease affecting which target do these patterns indicate?
- homogenous
- fine speckled
- centromere
Homogenous:
- Most common. Mostly resting interphase cells. Bright cells are dividing cells - DNA has lined up down centre
- target dsDNA
- disease association; SLE (95%), autoimmune hepatitis, rheumatoid arthritis
Fine speckled:
Fine speckling of nucleus. Dividing cells show the outside of cell but nothing in the centre.
- target SS-A (Ro), SS-B (La) and through cytoplasmic staining – Jo-1
- disease association; Sjogren’s syndrome (95%), SLE (40%), Scleroderma (5%) and Polymyositis (20-40%)
Centromere:
Resting cells - spread out dotty pattern (one dot per chromosome), dividing cells = each dot lined up on centre, depending on the stage will either have 23 or 46.
- target CENT A-E
- disease association; CREST (60%) and Raynaud’s
What are heterophile antibodies?
Human antibodies with multi specificity
Against poorly defined antigens
Weak antibodies produced during process of antibody diversity
Produce false positive staining - usually streaks or all over green fluorescence
What does monkey ABO cross-reactivity involve?
Humans and monkeys possess 97-98% of identical epitopes and share similar structural characteristics of the organs themselves.
Disadvantage - similarity in the epitopes can lead to ABO blood group cross-reactivity between antigens in monkey tissue and human blood
This can lead to high background staining making interpretation difficult
Can be overcome by using an antibody neutralising reagent which binds to, and inhibits the patient ABO-related antibodies
What are the advantages and disadvantages of automating immunofluorescence?
Advantages:
- Free up staff time
- Consistency in reading
- Can decide what your cut-off threshold is
- Can link to LIMS – reduce transcription errors
- Image saved – can re-check at any point in the future
Disadvantages:
- May loose the reading skills
- Slower than reading manually
- Limited to the number of slides that can be loaded to read
- Not all slides can be read by this method
- Must use the slides from the same manufacturer as the reading system
What are the different types of patterns?
Homogenous Granular/fine speckled Centromere M2 mitochondria Few nuclear dots pattern Nuceolar Gastric parietal cell pattern
