Diagnostics Flashcards
3 tests done if have fever
Liver function test- albumin, total bilirubin , alkaline phosphatase, alanine amino transferase
Urea and electrolytes- sodium, potassium, urea and creatinine
Blood glucose
Different colour tubes for blood
Red- contains nothing- Urine and electrolytes, thyroid, liver function
Yellow- gel to speed up clotting- helpful to separate serum and RBC- for urine and electrolytes, thyroid, liver function
Purple- contains potassium EDTA- anticoagulant- preserves for 6 hours- HBA1c
Grey- contain fluoride oxalate - kills RBC- blood glucose measurements
Green- contain heparin
Blue- contain citrate(anticoagulant- removes calcium)- use to see if someone can make clotting factors- add calcium
HB1AC
Measurement in diabetes
Haemoglobin is glycated so glucose sticks to it
If perform electrophoresis in diabetes HbA is blurred because glucose sticks to it randomly making it larger
Glycation takes 3 months so can see if diabetic patient has been monitoring properly
Serum vs plasma
Plasma has clotting factors in it
Measuring glucose
RBC consume glucose
So longer left fewer glucose
Fluoride oxalate- prevents RBC using glucose
When to contact chemical pathologist
When you want the sample to be rapidly centrifuged out of hours
When you want to measure labile hormones such as insulin (breaks down quickly)
When you urgently need CSF glucose and protein to be measured (e.g. in meningitis – emergency)
Renal function
Creatinine- marker of glomerular filtration rate
Urea- levels rise in dehydrated patients
Liver enzymes and protein
Tiny amount leaks into blood
In liver disease- more leak into blood e.g ALT
ALP (alkaline phosphate), AST, gamma GT, ALT(alanine amino transferase)
Bilirubin and albumin
Cardiac Enzymes- use in blood test and what are they
Enzymes present in heart muscle
During heart attack- muscle damaged and they leak into blood
Troponins (if this is high- definitely something wrong)
Creatiniine Kinase
Aspartate amino transferase (AST)
Lactate dehydrogenase (LDH)
What can be detected in the lab that’s useful in identifying viruses
The virus itself and components
Protein components
Genetic components
Host response- antibodies
Diagnostic methods of virology
Antibody detection Antigen detection Genome detection Serotyping Genom sequencing Quantification of antibody or antigen or genome
Limitations of laboratory tests
Sensitivity- test ability to correctly identify positive samples (less false negative)
Specificity- test’s ability to correctly identify negative sample (less false positive)
Typical samples used to contain virus
- Throat swab, Nasopharyngeal aspirate (NPA), bronchoalveolar lavage (BAL), ET secretions – for detection of respiratory viruses by (IF or) PCR
- Stools – for rotavirus, adenovirus & norovirus antigen detection (EIA) or PCR
- Urine – for BK virus & adenovirus PCR
- CSF – for herpes viruses and enteroviruses PCR
- Blood (clotted) - for serology (antibody detection)
- Blood (EDTA) - for PCR / viral load testing
- Saliva – for serology &/or PCR (eg measles
Use of serology
HIV Hepatitis A IgM and IgG HBV Measles, mumps, rubella IgM and IgG Parvovirus B19 IgM and IgG
IgM versus IgG results
IgM is a marker for acute infections or recent
IgG in absence of IgM indicates infection in the past. or immunisation
HIV serology
All reactive samples undergo confirmatory testing in a second assay to exclude non-specific reactivity (false positive)
Confirmed positives undergo typing (1 or 2)
Serology allows detection before AB get to detectable level
Highly automated, allowing to test more sample, quickly and cheaply
Virus isolation in cell culture
Time consuming and expensive- only performed in specialised labs
Used for phenotype susceptibly testing
Electron microscopy in virology diagnostics- what samples used
Only used in limited labs, only EM used
Used in samples of stools and vesicle fluids
Use of immunofluorescence in virus detection
Sometimes use for direct detection of viral antigens
Rapid and inexpensive but dependent on skilled technician and quality of sample
Antibody avidity testing- in IgM
IgM tests usually show low specificity- give rise to high false positives
In acute phases- avidity is low
Maturation causes increased avidity over time
So if avidity is high shows past infection, is avidity is low shows acute/recent infection
Respiratory tract infection- samples
- Throat swab +/- nose swab
- Nasopharyngeal swab
- Nasopharyngeal aspirate (NPA)
- Bronchoalveolar lavage (BAL)
- Endotracheal tube (ET) secretions
Testing of respiratory tract infections
Multiplex PCR assay (multiplex meaning can test for several viruses per tube)
Investigating CNS diseases- meningitis and encphalitis
CSF is used as the sample
Investigating diarrhoea and vomiting
Stools preferred sample
Use PCR
Enteric viruses cause diarrhoea and vomiting
Types of enteric viruses
Norovirus, rotavirus, adenovirus
PCR use in both DNA and RNA viruses, and its steps
If RNA virus- make ds-DNA copy
Achieved through reverse transcription
Denaturation (95oC)
Annealing (50oC)
Primer extension (72oC)
Real time PCR
Provides objective, computerised read out of results
Shows DNA quantitatively
Common diagnostic technique for bacteria
Culture of sterile sites (blood/CSF) and non sterile sites (urinary tract/bowel/skin)
Serology – looking for an amounted immune response to infection
Molecular Techniques – e.g. PCR – looking for presence of bacterial DNA/RNA
Antimicrobial Susceptibility Testing
Blood cultures of bacteria- what happens
Blood gets put into 2 different bottles- anaerobic and aerobic
Blood incubated and bacteria allowed to multiply
When they reproduce they produce Co2- causing pH and colour change in disc at bottom of tube
This change flags alarm on machine
Positive blood cultures of bacteria
Blood is removed and put onto different agar plates
Most grow well on blood/chocolate (haemolysed blood) agar, but gram negative grow well on MacConkey agar
Presence on MacConkey’s produces colour change
There is also neomycin agar
Determining bacteria through cell wall
Use gram stain
Gram+ purple due to thick peptidoglycan wall retaining stain
Gram- pink due to thin peptidoglycan wall between membranes
Detecting between staphylococci
Coagulase test
If positive- Staphy aureus
If negative- usually not problem such as commensals
Determining between Streptococci
If use up all blood (haemolysis)- beta haemolytic
If produce green tinge- alpha haemolytic
If non haemolytic- could be commensal strain of enterococci
Gram negative bacilli in blood
Should worry about septic shock
Have outer membrane, produce toxins and cause shock
Investigating patient’s stool sample
Stool has to be fresh
Cultured on agar plates
Only Salmonella, Shigella and Campylobacter are routinely looked for
Clostridium difficile- toxin detection or PCR for toxin gene as can’t culture
Sensitivity testing for bacteria
Look at point at which bacteria is resistant or sensitive to specific conc of drug
Minimum Inhibitory Concentration
Perform a doubling dilution – with decreasing antibiotic at each stage. You are looking for the point at which bacteria start growing – this is the MIC.
Control at end with no antibiotic
Beta-lactamases
Enzymes that bacteria have to help survive against environmental competitors
Seroconversion in bacteria- and limitations
Initial exposure- IgM response early on
Rise later on if exposed again
IgG response happens later on
However may get negative serology result for IgG since infection may be present but sample is sent off too early. Therefore important to repeat tests 2-4 weeks
Try send sample before antibiotic use
Histopathologist vs cytopathologist
Histo- interested in tissues
Cyto- interested in cells
Histopathology- biopsies, what are you looking at
See if inflammaed
See if malignant- type of cancer too
Histopathology- resection specimens- use?
Tells us how far disease has spread
Whether invaded different parts of body- or entered lymph nodes
Histopathology- frozen sections- what can be determined by them
A surgeon can open up a patient, take a tissue sample, and give it to the histopathologists
The histopathologists determine whether any masses are benign or malignant
If there is malignancy, the surgeons do a full resection of the tumour to remove it from the body
What is done to of post-mortem samples in histopathology
Samples are fixed in foramen- cross link protein preventing decomposition
Then put in paraffin wax- allowing them to cut very thin sections
Stained with haematoxylin and eosin
Other stains include gram stain and Ziehl-Neelsen stain (for tuberculosis)
pecific antigens can be identified using antibodies (immunohistochemistry)
Fine-needle aspiration
Cytopathologists insert small needle to obtain sample
Then look at cells
What is attached to antibodies’ constant part in tests
- Attach enzymes e.g. peroxidase, alkaline phosphatase
- Attach fluorescent probes e.g. dyes, beads of different sizes
- Attach magnetic beads e.g. purification of cell types
- Attach drugs e.g. kadcyla, anti-HER2 linked to emtansine
Generating monoclonal antibodies
2 different cell types fused together- making hybridoma
One of the cell types- produce antibodies (B cells, spleen) of interest- but limited in capacity for division
Other cell type- myeloma cells- can grow indefinitely
When fused- immortal cells that produce antibody of interest
In the medium containing the HAT enzyme- only fused cells are able to survive
Production of antibodies using recombinant DNA technology
Immobilise antigen we want antibodies against
Library of bacteriophages containing different antibody genes poured onto same plate
Antibodies that bind will stick, others washed away
End up with single bacteriophage with optimum specificity
Use of manufactured antibodies- therapeutic
- Prophylactic protection against microbial infection e.g. IVIG, synagis (anti-RSV)
- Anti-cancer therapy e.g. anti-HER2
- Removal of T-cells from bone marrow grafts – Anti-CD3
- Block cytokine activity e.g. anti-TNF
If -omab- mouse
- imab- chimeric or partly humanised
- umab- fully humanised
Use of manufactured antibodies- diagnostic
- Blood group serology
- Immunoassays – hormones, antibodies, antigens
- Immunodiagnosis – Infectious diseases, Autoimmunity, Allergy (IgE), Malignancy (myeloma)
ELISA
The antigen immobilised. An antibody against the antigen is added. This antigen has a reported molecule on it (e.g. enzyme). We wash away the unbound antibody – if there isn’t any antigen, there is no bound antibody. We add the substrate, and see the development of the colour (which can easily be measured by a spectrophotometer). The level of the colour gives a quantitative measure of the amount of antigen
Rapid testing- with antibodies
Rapid testing (dipstick tests, strip tests) use antibodies to develop coloured lines. Sample pad absorbs sample- contains antibodies against sample
First strip- contains antibody against thing trying to measure
Control strip after first strip- containing anti-antibody
To show sample has reached this region
Immune complexes
Depending on the ratio of antibody to antigen, you can get large immune complexes, or small ones.
Large complexes good at activating complement, neutrophils and platelets
Small don’t activate cells efficiency, but once immobilised on cell membranes- efficient at activating
Immunodeficiency tests
Serum Immunoglobulin levels
- (Serum electrophoresis /ELISA/Nephelometry)
Specific Antibodies (ELISA)
- Protein antigens – Tetanus & Haemophilus
- Polysaccharides antigens – Pneumococcus
Lymphocyte subsets (Flow Cytometry)
Serum electrophoresis
The electrophoresis results show a massive band of albumin (the most abundant protein in serum). The gamma-globulin region contains most of the antibodies.
Differential to healthy control can show active immune response and perhaps myeloma
Lymphocyte subset
Use antibodies against specific maker- with fluorescent dye Passed through flow cytometer- one cell at a time which detects fluorescence * CD3+ T cells – pan T cell marker * CD4+ T cells – T helper/cells * CD8+ T cells – cytotoxic T cells * CD19+ B cells * CD56+ Natural Killer (NK) Cells
Can quantify number of cells against/in certain disease e.g CD4+ against HIV
HIV positive patient response
Measure viral load and CD4 count
if CD4+ falls below 500/microlitre- treated with ARV therapy
Can’t let levels get too low or else common infections can be fatal
