Laboratory Techniques: Investigation Methods PDF

2.4 INVESTIGATION Nucleic acids: - In situ hybridisation - PCR - Electrophoresis (N + S blot) - Mass Spectrometry (DNA + RNA sequencing) Protein: - Immunostaining - Flow cytometry - Fluorescence-activated cell sorting - Enzyme-linked immunosorbent assay - Electrophoresi...

2.4 INVESTIGATION Nucleic acids: - In situ hybridisation - PCR - Electrophoresis (N + S blot) - Mass Spectrometry (DNA + RNA sequencing) Protein: - Immunostaining - Flow cytometry - Fluorescence-activated cell sorting - Enzyme-linked immunosorbent assay - Electrophoresis (W blot) - Mass spectrometry (proteomics) Immunocytochemistry - Anatomically visualise location of specific protein/antigen by binding antibodies Flow cytometry: ** Cells in a Fluid: A sample of cells is suspended in a liquid.** ** Flow in Single File: The cells are directed to pass through a narrow stream, one at a time.** ** Laser Detection: As each cell passes through a laser, it scatters light. Specialized detectors measure:** - **Size (using forward scattered light).** - **Granularity/complexity (using side scattered light).** ** Fluorescent Labels: If the cells are stained with fluorescent markers (tags that bind to specific proteins or molecules), the laser excites these markers, and the emitted light is detected.** ** Data Analysis: The signals are processed to determine the properties of the cells, such as their size, shape, and the presence of specific markers.** - Can be used to measure cells, surface expression, reg of TLR, adaptor binding protein - Sort cells by type, marker expression - Bead assay = particles coated in specific molecules to bind to protein that mimic interaction - Internilisation = intracellular expression **FAC:** -  **Flow Cytometry**: Like regular flow cytometry, FAC passes cells one by one through a laser beam and detects their properties using light scattering and fluorescence. -  **Sorting**: After analyzing each cell, FAC uses tiny electric charges to sort the cells based on their features (like size, shape, or specific fluorescent markers). -  **Collection**: The sorted cells are directed into separate containers, so you end up with groups of cells that have specific characteristics. ELISA: - Measures protein expression/secretion -\> antibody specific binding - Additional prep techniques (centrifugation, fractionation, lysis...etc) = internal protein detection in suspension - Multiplexing techniques = multiple targets detected in small sample volume - Quantification calculated by comparison to known standard curve - **Direct ELISA:**\ PROS: minimum procedure; avoids cross-reactivity from secondary antibody. - CONS: requires labeling of all primary antibodies - high cost; not every antibody is suitable for labeling. - **Indirect ELISA:**\ PROS: secondary antibodies are capable of signal amplification; many available secondary antibodies can be used for different assays; unlabeled primary antibodies retain maximum immunoreactivity. - CONS: cross-reactivity may occur. - **Sandwich ELISA:**\ PROS: sensitive, high specificity, antigen does not need to be purified prior to use. - CONS: antigens must contain at least two antibody binding sites. - **Competitive ELISA:**\ PROS: crude or impure samples may be used, high reproducibility. - CONS: lower overall sensitivity and specificity. - **Multiplex:** - PROS: high specificity, short run time, economical sample usage, multiple targets in one run. - Cons: EXPENSIVE! PCR: - DNA/RNA amplification - Sections of DNA/RNA using complimentary primers for analysys - Denaturing -- annealing -- extension - Reverse transcription -- quantitative polymerase chain reaction = isolation of mRNA -\> reverse transcribed into complementary DNA -\> transcription binding dye - Limitations = contamination, non-specific primer binding -\> amplification of multiple products Electrophoresis: - Sorting molecules by size + charge - Sample put on gel matrix -\> agarose for DNA, polyacrylamide for protein Blotting techniques: - Transfer of separated nucleic acids/proteins onto membrane - Western blotting = labelled antibodies detects target molecule - Labelled nucleic acid probes recognise complimentary DNA (southern) or RNA (northern) - Detection = luminescence, fluorescence or radioactivity Mass spectrometry: - Sorts molecules by mass to charge ratio - Protein analysis = proteomic analysis -\> proteomic profiling of whole cell, intra and extracellular protein - Labelling = specific targeting - Can provide info on protein folding, structure + function - Limitations: expensive, lots of irrelevant data, sensitive to poor prep and isolation techniques Microscopy:- fluorescence in-situ hybridisation CHILDHOOD DISEASE Herpes Simplex Virus 1 + 2 -- Human alphaherpesvirus 1: - Aka cold sores, HSV 1+ 2 - Transmission: contact w herpetic lesions, mucosa surface + oral and genital secretions -\> children can indicate sexual abuse - Incubation: 4 days (2-12 days) - Signs + symptoms: initial infection can include mild pyrexia, myalgia, anterior cervical lymphadenopathy. Difficulty eating, painful recurring blisters and ulcers, vesicles on hard palate, fauces. Herpatic lesions on anterior part of mouth - Treatment: +/- 2 weeks to heal. Oral therapy w acyclovir - Prevention: avoid contact, reschedule active lesions - Complication: immunodeficiency = more severe symptoms + more recurrences. Meningocenphalitis, encephalitis, keratis, neurological issues with neonates Varicella: - Transmission: direct contact, broken blisters, airborne droplets - Incubation: 10-20 days - Signs + symptoms: rash, pruritus, oral unilateral vesicles followed by ulceration (can devitalise teeth/bone necrosis), mild pyrexia. Macules -\> papules -\> vesicles -\> crust - Treatment: calamine lotion, antipyretic, acyclovir for severe cases. Benzydamine spray/mouthwash - Prevention: avoid contact w pt, vaccination - Complications: bacterial infection of lesion, scarring, conjunctivitis, pneumonia, meningitis, encephalitis, myocarditis, Reye's syndrome Infective mononucleosis: human gammaherpesvirus 4 - Transmission: infected saliva, most common in teens - Incubation: 4-7 weeks - Signs + symptoms: exudate pharyngitis, tonsilitis, lymphadenopathy, pyrexia, cephalgia, myalgia, malaise, anorexia, hepatitis, enlarged spleen and liver - Treatment: symptom relief, antipyretic, fluid intake - Prevention: avoid contact - Complications: spleen and liver enlargement, lymphoma, prolonged fatigue. Malaise - Infects B lymphocytes -\> cytotoxic T respond -\> downey cells Epidemic parotitis -- mumps orthorubulavirus: - Transmission: droplets and secretions - Incubation: 15-25 days - Signs and symptoms: uni/bilateral parotitis, myalgia, anorexia, malaise, cephalgia, mild pyrexia - Treatment: relieving symptoms - Prevention: vaccination - Complications: orchitis (post-pubertal males), sterility (males), pancreatitis, facial paralysis, deafness, cerebellar ataxia and polyneuritis, meningoencephalitis, other systemic inflammations Enteroviral vesicular stomatitis with exanthem -- enterovirus spp. - Transmission: droplets + faeces - Incubation 6 days - S&S: pyrexia, sore throat, malaise, loss of appetite -\> Later yellow/red oral lesion, vesicles in hands and feet - Prevention: avoid contact, good hygiene, surface disinfecting - Complication: rare -- viral meningitis, encephalitis, flaccid aralysis, fingernail + toenail loss Measles -- morbillivirus hominis: - Transmission: droplets - Incubation: 10-12 days - S&S: pyrexia, conjunctivitis, lymphadenopathy, anorexia. Kolpik's spots -\> cluster of white lesions in molar area - Treatment: symptom relief, antibiotics w secondary bacterial infection - Prevention: vaccination - Complication: Otitis media, pneumonia, croup, convulsions and encephalitis, subacute sclerosing, encephalitis, death Respiratory Synsytial Virus (RSV) -- orthopneumovirus hominis: - Transmission: droplets, direct contact, fomites - Incubation: 2-8 days - S&S: common cold, nasal congestion. Rhinorrhoea, sternutation, fatigue, pyrexia - Treatment: hospitalisation. Fluids, erythromycin, maintaining airway - Prevention: vaccination Scarlet fever -- streptococcus pyogenes (group A Streptococcus): - Transmission: normal flora of oropharynx, droplets - Incubation: 12hours -- 7days - S&S: pyrexia, chills, sore throat, cephalgia, vomiting, abdominal pain, pruritus, tachycardia, chite coating on tongue, dequamated tongue, swollen tonsils - Treatment: antipyretic, antibiotics (penicillin) - Prevention: long term antibiotics Whooping cough -- Bordetella pertussis: - Transmission: droplets, direct contact - Incubation: 7-10 days - S&S: common cold, pyrexia, tachycardia. rapid breathing, malaise, dry cough lasting more than a minute, anxiety, vomiting - Treatment: hospitalisation, fluids, erythromycin, maintaining airway - Prevention: vaccination Dermatophytosis -- dermatophytosis spp.: - Transmission: contact with infected soil or animals - Incubation: N/A - S&S: round ring shaped, silvery and scaly lesion. Edge raised red. Ring spreads outwards and may blister and ooze, scalp infection may cause some bald patched and lymphadenopathy - Treatment: topical corticosteroids, topical antifungal cream (miconazole), iodine shampoo, oral griseofulvin - Prevention: check pets frequently, don't share hygiene products, handwashing Meningtitis: **Cause by meningococcus, Neisseria meningitidis, streptococcus pneumoniae, heamophilus influenzae, streptococcus agalactia (group B strep)** - Transmission: respiratory secretions, droplets - Types: viral bacterial, fungal, parasitical - S&S: stiff neck, pyrexia, vomiting, cephalgia, rash that doesn't blanch, photophobia, unresponsiveness or drowsiness, seizure, confusion/altered mental state, infants arching back - Treatment: diagnosis usually requires lumbar puncture, antibiotics, respiratory support, treating blood pressure, would care - Prevention: vaccination Sepsis: - Transmission: not contagious - Increased risk: babies \>1, 75+, diabetes, immunocomprimised, surgery recovery, illness, post-birth - S&S: blue, pale, blotchy lips, rash don't blanche, difficulty breathing, fast breathing, vomiting, no urination, pyrexia - Treatment: ICU, ventilation, surgery - Prevention: vaccination, clean wounds, antibiotics, handwashing