W1: Fundamentals of Clinical Laboratory Techniques

Question 1

what are the 4 elements of clinical context?

Answer 1

diagnosis
aetiology
monitoring
guided therapy (genetic susceptibility)

Question 2

Cobas ISE module

Answer 2

ISE: “Ion Selective Electrode”
V specific method, uses potentiometric measurement to determine conc of Na+, K+ & Cl-

Sample types: serum, plasma, urine & fluids
V quick test – 30 secs
Electrolyte measurement
Can process 1800 tests/hr

Question 3

principle of electrolyte analysis

Answer 3

ISE consists of an ion selective electrode & a ref electrode
Sample diluted & electrodes placed w/in sample
Ion selective electrode has mem thru which only ion of interest can pass
Ion exchange occurs across mem -> gen of an EP which reflects sample comp

Question 4

causes of electrolyte disturbances: low sodium

Answer 4

heart failure
head injury
medications e.g. diuretics

Question 5

causes of electrolyte disturbances: high sodium

Answer 5

inadequate water intake
diabetes insipidus
water loss e.g. diarrhoea, vomiting

Question 6

causes of electrolyte disturbances: low potassium

Answer 6

inadequate nutrition
vomiting/diarrhoea
medications e.g. diuretics

Question 7

causes of electrolyte disturbances: high potassium

Answer 7

spurious (old or haemolysed sample)
kidney dysfunction
Addison’s disease

Question 8

limitations of the Cobas ISE modules

Answer 8

indirect ISE method : serum (liquid portion of the blood) diluted in buffer before it encounters the ISE mem

sometimes prods erroneous results if patient has higher than normal levels of proteins &/or lipids

Question 9

what is the alternative type of ISE to indirect ISE?

Answer 9

direct ISE. Used in most point of care analysers. Doesn’t involve dilution of sample.

Question 10

+ves of an indirect ISE method

Answer 10

requires a v small amount ofsample
less expensive than direct ISE
has a large dynamic range

Question 11

Cobas ISE module example: pseudohyponatraemia

Answer 11

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Question 12

Roche Cobas c702 modules (Chemistry Unit)

Answer 12

Measure general chem assays e.g. urea, creatinine, LFTs, calcium, magnesium
Tests take 10 mins – can process 2000 tests/hr
Samples types: serum, plasma, urine, CSF, stool samples & fluids
Uses photometric tech to measure analytes.

Photometric assays: organic & inorganic compounds in solution measured by determining absorbance of wavelengths of light
When light enters photometer, hits a diffraction grating: splits light into diff wavelengths. These wavelengths reflected onto fixed array of 12 photodiodes
Each photodiode mounted into fixed position & detects light at an individual wavelength
Analytes measured: creatinine (kidney function), bilirubin (liver function),glucose, magnesium

Question 13

Roche Cobas c702 modules (Chemistry Unit) example: glucose hexokinase reaction

Answer 13

Reaction -> change of absorption at 340nm

Change in absorption measured & is prop to glucose conc

Question 14

limitations of the chemistry module

Answer 14

Limitations based around interferents -> changes in wavelength

Interferents affect sample integrity & can incl drugs, dyes, high levels of proteins, lipidsor cells in sample

Measurements also affected by clots/air bubbles

Sample integrity monitored by:
- Detection system for clots/bubbles & liquid level
- Measurement of serum indices

Question 15

serum indices

Answer 15

Measurements performed on every sample analysed on chemistry module
Absorbance measurements of diff wavelengths of light used to identifysamples thatare icteric, haemolysed & lipaemic
These interferents have variableeffects on measurement of diff analytes
If indices measurement breaches a threshold for a particular assay, ITsystem removes result bc not accurate

Question 16

Roche Cobas e801 modules (Immunoassay Unit)

Answer 16

Immunoassays: bioanalytical methods - quantitation of an analyte depends on reaction of an antigen (analyte) & an Ab
Most samples used are serum & plasma
9 mins - 32 mins
2000 tests/hr
Use electrochemiluminescence (ECL) tech

Question 17

sandwich immunoassay

Answer 17

Uses 2 Abs which bind to diff sites on analyte of interest
A capture Ab bound to solid surface. Sample added containing analyte of interest
A 2nd Ab added which recognises a diffepitope one theantigen, forming a sandwich
The 2ndAb has amolecular label whichforms part of an ECL reaction
Signal prop toconc of analyte in sample
Larger analytes e.g.TSH usesandwich assays

Question 18

competitive immunoassay

Answer 18

Onlyuses 1 Ab
Used forsmaller analytes e.g. Testosterone
Sample mixed w/ labelled analogue
Labelled analogue & analyte in sample compete for binding sites on thecapture Ab which is bound to a solid surface
Signal inverselyrelated to conc ofanalyte in sample

Question 19

limitations of immunoassay module

Answer 19

V high levels of analyte -> Hooke Effect in sandwich assays.
Ab prod expensive & prone to diffs btwn batches.
Cross reactivity ofsimilar analytese.g. some steroids & steroid medications
Some immunoassays use biotin-streptavadin interactions forbinding capture Ab to solid surface. Biotin supplementstaken by patient canbe source of interference.

Question 20

osmolarity

Answer 20

conc of dissolved particles w/in a solution

Question 21

osmometer

Answer 21

Serum & urine osmolalitymeasured toinvestigate cause of low serum [Na+] (hyponatraemia) or high serum [Na+] (hypernatraemia)
Osmolality used to investigate cause of high/low urine output or excessive thirst
Serum osmolalitymay be measured if ingestion of toxic alcohols e.g. methanol/ethylene glycol suspected
Osmometer instrument worksby principle of freezing point depression

Question 22

freezing point depression

Answer 22

Freezing point of a liquid lower when another substance dissolved in it
Sample injected into osmometer
Sample supercooled & agitated to cause crystallisation
Resulting heat of fusion  sample temp to rise to a plateau equilibrium temp (measured using high precision thermistors)
Used to calc osmolality of sample

Question 23

what is a sweat chloride analyser used to measure?

Answer 23

Measurement of chloride in sweat is used in diagnosis of CF
Patients w/ CF have higher levels of Cl- in sweat than unaffected individuals
This feature independent of genotype

Question 24

how is sweat collected

Answer 24

Localised sweating induced on surface of arm using electrical stim
Sweat collected in macroduct sweat collector

Question 25

how does a sweat chloride analyser work?

Answer 25

Works by Coulometric titration
2 silver electrodes dipped into a working solution of an acid buffer + stabiliser. This solution contains no silver ions, so the indicator current is brought to an end-point
When sweat sample pipetted in, indicator current drops & silver ions released by anode until all the Cl- are precipitated out as AgCl. This restores original [AgCl]
Period of flow of this current is prop to the sweat [Cl-]

Question 26

limitations of sweat chloride analysis

Answer 26

Some patients receive borderline results e.g.newborn screening/genotype +ve but inconclusive sweat test result
Method is manual – requires accurate pipetting & manual transcription
Sweat collection can take 30 mins
Can be difficult to obtain sufficientsweat from newborn

Question 27

spectrophotometer used to test for…

Answer 27

UvikonXL Spectrophotometer used to run an assay: CSF Xanthochromia
After a subarachnoid haemorrhage (SAH) blood leaks into cerebralventricles & CSF
Xanthochromia = yellow discolouration of CSF caused by release of oxyHb from disintegrating RBCs &subsequent conversion to bilirubin

Question 28

how does spectrophotometer work?

Answer 28

CSF collected via a lumbarpuncture
SP scans CSF sample using range of wavelengths to determine absorbance peaks
For Xanthochromia, instrument programmed to detect peaks demonstrating presence of oxyHb and bilirubin (breakdown products of blood)

Question 29

limitations of xanthochromia

Answer 29

Certain drugs, e.g. antibiotic doxycycline, can -> interferingpeaks
Manual method requiring specialist training
Previous lumbar punctures performed during last 2 weeksmay introduce traces of blood
Bilirubin breaks down in presence of light- important to protect sample from light
Test not valid until 12hrs after the poss event (takes 12hrs for blood to break down into bilirubin &oxyHb)

Question 30

name 6 diff analysers used in clinical biochem lab

Answer 30

Cobas ISEs
Cobas c702s (Chemistry Unit)
Cobas e801s (Immunoassay Unit)
Osmometer
Sweat chloride analyser
Spectrophotometer

Question 31

which 3 analysers are automated?

Answer 31

Cobas ISEs
Cobas c702s (Chemistry Unit)
Cobas e801s (Immunoassay Unit)

Question 32

which 3 analysers are manual?

Answer 32

Osmometer
Sweat chloride analyser
Spectrophotometer