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Diagnostics Flashcards

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1
Q

3 tests done if have fever

A

Liver function test- albumin, total bilirubin , alkaline phosphatase, alanine amino transferase
Urea and electrolytes- sodium, potassium, urea and creatinine
Blood glucose

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2
Q

Different colour tubes for blood

A

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

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3
Q

HB1AC

A

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

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4
Q

Serum vs plasma

A

Plasma has clotting factors in it

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5
Q

Measuring glucose

A

RBC consume glucose
So longer left fewer glucose
Fluoride oxalate- prevents RBC using glucose

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6
Q

When to contact chemical pathologist

A

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)

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7
Q

Renal function

A

Creatinine- marker of glomerular filtration rate

Urea- levels rise in dehydrated patients

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8
Q

Liver enzymes and protein

A

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

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9
Q

Cardiac Enzymes- use in blood test and what are they

A

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)

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10
Q

What can be detected in the lab that’s useful in identifying viruses

A

The virus itself and components
Protein components
Genetic components
Host response- antibodies

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11
Q

Diagnostic methods of virology

A 
Antibody detection
Antigen detection
Genome detection
Serotyping
Genom sequencing 
Quantification of antibody or antigen or genome
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12
Q

Limitations of laboratory tests

A

Sensitivity- test ability to correctly identify positive samples (less false negative)

Specificity- test’s ability to correctly identify negative sample (less false positive)

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13
Q

Typical samples used to contain virus

A
  • 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
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14
Q

Use of serology

A 
HIV
Hepatitis A IgM and IgG
HBV
Measles, mumps, rubella IgM and IgG
Parvovirus B19 IgM and IgG
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15
Q

IgM versus IgG results

A

IgM is a marker for acute infections or recent

IgG in absence of IgM indicates infection in the past. or immunisation

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16
Q

HIV serology

A

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

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17
Q

Virus isolation in cell culture

A

Time consuming and expensive- only performed in specialised labs

Used for phenotype susceptibly testing

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18
Q

Electron microscopy in virology diagnostics- what samples used

A

Only used in limited labs, only EM used

Used in samples of stools and vesicle fluids

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19
Q

Use of immunofluorescence in virus detection

A

Sometimes use for direct detection of viral antigens

Rapid and inexpensive but dependent on skilled technician and quality of sample

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20
Q

Antibody avidity testing- in IgM

A

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

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21
Q

Respiratory tract infection- samples

A
  • Throat swab +/- nose swab
  • Nasopharyngeal swab
  • Nasopharyngeal aspirate (NPA)
  • Bronchoalveolar lavage (BAL)
  • Endotracheal tube (ET) secretions
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22
Q

Testing of respiratory tract infections

A

Multiplex PCR assay (multiplex meaning can test for several viruses per tube)

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23
Q

Investigating CNS diseases- meningitis and encphalitis

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A

CSF is used as the sample

24
Q

Investigating diarrhoea and vomiting

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A

Stools preferred sample
Use PCR
Enteric viruses cause diarrhoea and vomiting

25
Types of enteric viruses
Norovirus, rotavirus, adenovirus
26
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)
27
Real time PCR
Provides objective, computerised read out of results | Shows DNA quantitatively
28
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
29
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
30
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
31
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
32
Detecting between staphylococci
Coagulase test If positive- Staphy aureus If negative- usually not problem such as commensals
33
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
34
Gram negative bacilli in blood
Should worry about septic shock | Have outer membrane, produce toxins and cause shock
35
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
36
Sensitivity testing for bacteria
Look at point at which bacteria is resistant or sensitive to specific conc of drug
37
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
38
Beta-lactamases
Enzymes that bacteria have to help survive against environmental competitors
39
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
40
Histopathologist vs cytopathologist
Histo- interested in tissues | Cyto- interested in cells
41
Histopathology- biopsies, what are you looking at
See if inflammaed | See if malignant- type of cancer too
42
Histopathology- resection specimens- use?
Tells us how far disease has spread | Whether invaded different parts of body- or entered lymph nodes
43
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
44
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)
45
Fine-needle aspiration
Cytopathologists insert small needle to obtain sample | Then look at cells
46
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
47
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
48
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
49
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
50
Use of manufactured antibodies- diagnostic
* Blood group serology * Immunoassays – hormones, antibodies, antigens * Immunodiagnosis – Infectious diseases, Autoimmunity, Allergy (IgE), Malignancy (myeloma)
51
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
52
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
53
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
54
Immunodeficiency tests
Serum Immunoglobulin levels * (Serum electrophoresis /ELISA/Nephelometry) Specific Antibodies (ELISA) * Protein antigens – Tetanus & Haemophilus * Polysaccharides antigens – Pneumococcus Lymphocyte subsets (Flow Cytometry)
55
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
56
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
57
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

MCD (2 decks)

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