LABS; 1, 2, 3, 4 and 5 - Diagnostics - Intro and chemical pathology labs, Virology lab, Bacteriology lab, Histopathology and cytopathology lab, Antibodies as diagnostic tools/Case summary Flashcards
How can you diagnose a virus?
History, physical examination and lap tests (non-specific and virological)
What can you detect in the lab?
Infectious virus (virus isolation and EM), Protein components (antigens) of the virus, Genetic components of the virus -> quant/qual tests available; host response (antibody/cell responses)
What are the diagnostic methods used in the lab?
Cell culture, electron microscopy, Antibody detection (serology - EIA), antigen detection (Immunofluorescence, enzyme immunoassay - EIA), genome detection (PCR), quantification of Ab/Ag, serotyping, quantification of genomes (viral load - essential for diagnosis and monitoring of HIV, HBV, HCV, and also for CMV and EBV in immunocompromised), genome sequencing (genotyping, antiviral resistance testing)
What are the limitations of lab tests?
All assays give rise to false negative and false positive results -> sensitivity (tests ability to correctly identify positive samples); specificity (tests ability to correctly identify negative samples)
Where are the typical samples taken?
Throat swab, Nasopharyngeal aspirate, bronchoalveolar lavage, ET secetion for resp viruses by PCR; Stools for rotavirus, adenovirus and norovirus Ag detection or PCR; Urine for BK virus and adenovirus by PCR; CSF for herpes viruses and enteroviruses by PCR; Blood (clotted) for serology Ab detection; Blood (EDTA) for PCR/viral load testing; saliva for serology and PCR (measles)
What serology is looked for?
How is IgM different from IgG?
How do you carry out Ab avidity testing?
What tests do the chemical pathology lab carry out?
LFT, Urea and Electrolytes, blood glucose
When collecting blood what should you do?
Use the correct tube; label the tube with patients details
What is inside the tubes with coloured lids - red, yellow, purple, grey?
Red - nothing; Yellow - gel to speed up clotting; Purple - potassium EDTA to prevent blood clotting and preserve cells Grey - fluoride oxalate (poison) which kills RBC so they don’t take up the glucose in the blood
What would you collect in each type of tube?
U+E serum in Y/R; glucose plasma in grey; HBA1c plasma in purple; TFT serum in Y/R; LFT in yellow/red
What is the difference between serum and plasma?
No anticoagulant, Blood clots using up clotting factors and then clot can be removed = serum; Anticoagulant, EDTA or heparin, clotting factors unused and blood separated into red cells and plasma = plasma
How do we test for clotting factors?
Citrate in blue tube chelates Calcium which stops the blood from clotting; time how long it takes to coagulate
How do you measure glucose?
Grey tube -> Fuoride oxalate prevents red cells from using glucose as it is a poison
What other components can be tested for in the blood?
Calcium and phosphate, markers of liver function (liver enzymes should be in the liver but if they leak then there is a problem, and some enzymes are specific to certain diseases - akaline phosphatase, AST, ALT, GGT), hormone assays, glucose
What causes low Na and high K?
Low Na is due to dehydration, K due to kidneys not functioning well
What is the meaning of Urea and Cr levels?
Both should be excreted by the kidney so measure renal function -> Cr should be excreted at a fixed rate; high urea is due to dehydration as it absorbs the water as well as the urea
What is the commonest cause of high K in the lab?
Haemolysed blood as RBC contain large amounts of K which leaks pout when haemolysed
What happens to urea and creatinine in renal failure?
Both rise but if creatinine is normal and urea rises then it is dehydration
What is creatinine?
Marker of glomerular filtration rate. If it is normal, then the GFR is normal. Very little is absorbed or secreted by the tubules.
What happens to urea levels when patient is dehydrated?
Levels rise but GFR stays the same to the end
What are the liver enzymes?
Present in liver and tiny amount leaks into blood -> liver disease more of these enzymes leak into the blood
Which liver function and enzymes are tested for?
AST, GGT, Albumin (synthesised in liver), bilirubin, alkaline phosphate, ALT
Why are cardiac enzymes are measured?
Present in heart muscle and during a heart attack, heart muscle is damaged, which causes enzymes to leak into the blood in large amounts -> then can check if heart attack actually happened
What cardiac enzymes are measured?
Troponins, creatine kinase, AST, lactate dehydrogenase
How would you test for HIV serology?
4th gen EIA (Ab and p24 Ag detection)’ all reactive samples undergo confirmatory testing in a second assay to excude non-specific reactivity (false positive), confirmed positives undergo typing HIV 1 vs 2; repeat blood sample +EDTA blood for HIV viral load required from all new +ves
When is virus isolation in cell culture used?
Rarely as it is slow, tme consuming, still useful for phenotypic antiviral susceptibility testing (HSV)
When is electron microscopy used?
Viruses too small to be seen by light microscopy, can be visualised using electron microscope -> sample types such as stools and vesicle fluids (rarely used)
When is immunofluorescence used?
Direct detection of viral antigens in clinical samples, used for typing and cell culture confirmation, rapid an inexpensive but subjective and very dependent on skill of technician and quality of sample
What tests would you do for a respiratory tract infection?
Throat swab +/- nose swab, nasopharyngeal swab, nasopharyngeal aspirate, bronchoalveolar lavage, endotracheal tube secretions -> use PCR
What kind of viruses cause respiratory tract infections?
Influenza, parainfluenza, RSV, rhinovirus, human metapneumovirus [HMPV], adenovirus, bocavirus, +/- coronavirus
Which CNS disease exist and how would you test for it?
CSF for PCR; Stools and throat swab for enterovirus detection; blood for serology and PCR for west nile or japanese encephalitis virus infection and other arboviruses
What are the different CNS diseases that can pop up in a clinical history?
How do you test D+V?
Stool (vomit as well), PCR or Ag detection assays -> Norovirus, rotavirus, adenovirus, sapovirus, astrovirus
What is PCR?
Polymerase chain reaction; method for amplifying specific RNA or DNA sequences
What are the common diagnostic techniques for bacteria?
Culture (sterile sites, non sterile sites), serology, molecular techniques, antimicrobial susceptibility testing
How would you diagnose Syphilis and Brucellosis?
Serology is used as it cannot be cultured
When would you use molecular techniques to diagnose bacteria?
Fecal pathogens
What are the common non-sterile sites?
How would you test the organisms in a sterile site?
Blood cultures -> use non-selecctive plates for sterile sites.
How do you test for Staphylococci?
Coagulase test
What is staphylococcus aureus?
Including MRSA -> causes severe infections (skin/soft tissue, endocarditis, osteomyelitis) -> skin commensals of low pathogenic potential whihc can infect prosthetic material causing line, pacemaker infections and endocarditis
Which MO’s cause diarrhoea?
Bacteria: Salmonella, shigella, campylobacter, E coli, C. difficile, Cholera; Parasites: Amoeba, Giardia, cryptosporidium; VIrus
How would you investigate bacteria in a stool sample?
Culture on agar plates -> only samonella, shigella and campylobacter looked for routinely; different pathogens have different culture requirements; clostridium difficile (toxin detection or PCR for toxin gene
How would you investigate parasites in stool samples?
Concentration and special stains
What is the minimum inhibitory concentration?
Test of moderately resistant bactericidal drug
How can you test for antibiotic resistance in bacteria?
MIC and disc diffusion
What do histopathologists use?
Biopsies, resection specimens, frozen sections, post-mortems
What do cytopathologists use?
Smears and fine needle aspirate
What do you look for in a biopsy?
Normal? Inflamed? Cancer?
What do you look for in a resection specimen?
How far has cancer spread? Is it all out?
What do you look for in a frozen section?
Rapid diagnosis -> is it cancer and is it all out?
What are the 2 types of post- mortem?
Hospital and coroner’s post-mortem
How are sections obtained?
Specimen must be properly labelled, fix in formalin, embed in paraffin wax, cut sections
What happens with the sections that are taken?
Stain (gram, ZN), identify specific Ag using Ab (immunohistochemistry), carry out molecular tests
When is cytopathology used?
Looking at individual cells not tissues ; usd for fine needle aspirations, used for cervical screening
How long does a histopathology result take to reach clinician?
Frozen section (30min), for biopsies (2-3d), resection specimens (5-7d)
What is Kaposi’s sarcoma?
HIV/AIDS defining disease, with immunocytochemistry for CD31 to show vascular tumour infiltrating collagen bundles
What can we attach to the constant part of the anti-body?
Enzymes, fluorescent probes, magnetic beads, drugs
Why are Ab used in diagnostics?
Unique specificity of Ab for their target Ag is the basis of many diagnostic tests -> Ab can be raised against almost any Ag including Ig from other species (anti-Ab)
What are the 2 types of Ab used?
Produced by patient (in AI disease, for defence against infection); manufactured Ab (antisera from immunised animals, monoclonal ab, genetically engineered Ab)
How do you generate monoclonal Ab?
How do you generate Ab using recombinant DNA?
x
What are the therapeutic uses of manufactured Ab?
Therapeutic: Prophylactic protection against microbial infection (IVIG, synagis), anti-cancer therapy (anti-HER2), removal of T-cells from bone marrow grafts (Anti-CD3), block cytokine activity (anti-TNFalpha)
What are the types of therapeutical monoclonal Ab?
-omab (mouse monoclonal; muronomab); -imab (chimeric or partly humanised, infliximab); -umab (palivizumab)
What are the diagnostic uses of manufactured Ab?
Blood group serology, immunoassays (hormones, Ab, Ag), immunodiagnosis (infectious diseases, AI, allergy, malignancy)
What is ELISA?
Enzyme linked Immunosorbent Assay
How do you rapidly test a substance?
What are signs and symptoms associated with immune complexes?
Inflammation/complement activation, serum sickness (complexes in circulation), immune complex glomerulonephritis, immune complex deposition at other sites (skin, joints, lungs)
How do you measure immunodeficiency?
What is serum electrophoresis?
Monoconal expansion of B cell (malignancy?), investigate for myeloma
What is flow cytometry?
How do you carry out the treatment for HIV?
What does the CD4 T cell count define in ART naive HIV-1 patients?
Extent of immune damage and predicts short term outlook
