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MCD > Diagnostics > Flashcards

Diagnostics Flashcards

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

A

CSF is used as the sample

24
Q

Investigating diarrhoea and vomiting

A

Stools preferred sample
Use PCR
Enteric viruses cause diarrhoea and vomiting

25
Q

Types of enteric viruses

A

Norovirus, rotavirus, adenovirus

26
Q

PCR use in both DNA and RNA viruses, and its steps

A

If RNA virus- make ds-DNA copy
Achieved through reverse transcription

Denaturation (95oC)
Annealing (50oC)
Primer extension (72oC)

27
Q

Real time PCR

A

Provides objective, computerised read out of results

Shows DNA quantitatively

28
Q

Common diagnostic technique for bacteria

A

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
Q

Blood cultures of bacteria- what happens

A

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
Q

Positive blood cultures of bacteria

A

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
Q

Determining bacteria through cell wall

A

Use gram stain
Gram+ purple due to thick peptidoglycan wall retaining stain
Gram- pink due to thin peptidoglycan wall between membranes

32
Q

Detecting between staphylococci

A

Coagulase test

If positive- Staphy aureus
If negative- usually not problem such as commensals

33
Q

Determining between Streptococci

A

If use up all blood (haemolysis)- beta haemolytic

If produce green tinge- alpha haemolytic

If non haemolytic- could be commensal strain of enterococci

34
Q

Gram negative bacilli in blood

A

Should worry about septic shock

Have outer membrane, produce toxins and cause shock

35
Q

Investigating patient’s stool sample

A

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
Q

Sensitivity testing for bacteria

A

Look at point at which bacteria is resistant or sensitive to specific conc of drug

37
Q

Minimum Inhibitory Concentration

A

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
Q

Beta-lactamases

A

Enzymes that bacteria have to help survive against environmental competitors

39
Q

Seroconversion in bacteria- and limitations

A

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
Q

Histopathologist vs cytopathologist

A

Histo- interested in tissues

Cyto- interested in cells

41
Q

Histopathology- biopsies, what are you looking at

A

See if inflammaed

See if malignant- type of cancer too

42
Q

Histopathology- resection specimens- use?

A

Tells us how far disease has spread

Whether invaded different parts of body- or entered lymph nodes

43
Q

Histopathology- frozen sections- what can be determined by them

A

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
Q

What is done to of post-mortem samples in histopathology

A

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
Q

Fine-needle aspiration

A

Cytopathologists insert small needle to obtain sample

Then look at cells

46
Q

What is attached to antibodies’ constant part in tests

A
  • 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
Q

Generating monoclonal antibodies

A

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
Q

Production of antibodies using recombinant DNA technology

A

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
Q

Use of manufactured antibodies- therapeutic

A
  • 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
Q

Use of manufactured antibodies- diagnostic

A
  • Blood group serology
  • Immunoassays – hormones, antibodies, antigens
  • Immunodiagnosis – Infectious diseases, Autoimmunity, Allergy (IgE), Malignancy (myeloma)
51
Q

ELISA

A

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
Q

Rapid testing- with antibodies

A 
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
Q

Immune complexes

A

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
Q

Immunodeficiency tests

A

Serum Immunoglobulin levels

  • (Serum electrophoresis /ELISA/Nephelometry)

Specific Antibodies (ELISA)

  • Protein antigens – Tetanus & Haemophilus
  • Polysaccharides antigens – Pneumococcus

Lymphocyte subsets (Flow Cytometry)

55
Q

Serum electrophoresis

A

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
Q

Lymphocyte subset

A 
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
Q

HIV positive patient response

A

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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