W1: Fundamentals of Clinical Laboratory Techniques Flashcards
what are the 4 elements of clinical context?
diagnosis
aetiology
monitoring
guided therapy (genetic susceptibility)
Cobas ISE module
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
principle of electrolyte analysis
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
causes of electrolyte disturbances: low sodium
heart failure
head injury
medications e.g. diuretics
causes of electrolyte disturbances: high sodium
inadequate water intake
diabetes insipidus
water loss e.g. diarrhoea, vomiting
causes of electrolyte disturbances: low potassium
inadequate nutrition
vomiting/diarrhoea
medications e.g. diuretics
causes of electrolyte disturbances: high potassium
spurious (old or haemolysed sample)
kidney dysfunction
Addison’s disease
limitations of the Cobas ISE modules
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
what is the alternative type of ISE to indirect ISE?
direct ISE. Used in most point of care analysers. Doesn’t involve dilution of sample.
+ves of an indirect ISE method
requires a v small amount ofsample
less expensive than direct ISE
has a large dynamic range
Cobas ISE module example: pseudohyponatraemia
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Roche Cobas c702 modules (Chemistry Unit)
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
Roche Cobas c702 modules (Chemistry Unit) example: glucose hexokinase reaction
Reaction -> change of absorption at 340nm
Change in absorption measured & is prop to glucose conc
limitations of the chemistry module
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
serum indices
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
Roche Cobas e801 modules (Immunoassay Unit)
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
sandwich immunoassay
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
competitive immunoassay
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
limitations of immunoassay module
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.
osmolarity
conc of dissolved particles w/in a solution
osmometer
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
freezing point depression
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
what is a sweat chloride analyser used to measure?
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
how is sweat collected
Localised sweating induced on surface of arm using electrical stim
Sweat collected in macroduct sweat collector
how does a sweat chloride analyser work?
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-]
limitations of sweat chloride analysis
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
spectrophotometer used to test for…
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
how does spectrophotometer work?
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)
limitations of xanthochromia
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)
name 6 diff analysers used in clinical biochem lab
Cobas ISEs
Cobas c702s (Chemistry Unit)
Cobas e801s (Immunoassay Unit)
Osmometer
Sweat chloride analyser
Spectrophotometer
which 3 analysers are automated?
Cobas ISEs
Cobas c702s (Chemistry Unit)
Cobas e801s (Immunoassay Unit)
which 3 analysers are manual?
Osmometer
Sweat chloride analyser
Spectrophotometer
