2: Clinical lab instrumentation

Question 1

Spectrophotometry

Answer 1

• Absorption of radiation of substance = absorption spectroscopy
• Advantages:
o Fast
o Sensitive to small amounts
o Simple operating methods
• Relies on selective absorbance of light by different samples

Question 2

Spectrophotometer

Answer 2

• Light source- tungsten lamp (360-920nm) or deuterium (220-360nm- UV range)
• Wavelength selector/filter- narrows range
• Monochromators- prisms/diffraction gratings diffract light towards sample where a second filter only allows specified wavelength to enter sample curvette
• Sample curvette- contains sample
• Detector- determines how much of the selected wavelength has been absorbed

Question 3

Flame photometry/atomic emission spectroscopy

Answer 3

• Atoms thermally excited
• Radiation detected
• Amount detected is indicative of the atoms concentration in the sample
• Differs from spectrophotometry&raquo_space; emission rather than absorption, usually used for pure metals (Na, K, Li)

Question 4

Fluorometry

Answer 4

• Ability of sample to absorb photon energy then re-emit photons with less energy
• 2 monochromators used- 1 before to isolate excitation light, 1 after to isolate emitted light

Question 5

Chromatography

Answer 5

• Detection of complex substances- drugs, hormones etc.
• Method of separating mixtures into component parts
• 1st phase = mobile (gas or liquid), 2nd phase = fixed (solid or liquid)
• Separation achieved during mobile phase
• Methods classified based on mobile method- i.e. liquid or gas chromatography
• Solid stationary phase based on absorption
• Liquid stationary based on divergence due to solubility

Question 6

Electrophoresis

Answer 6

•	Molecules separated by migration through an electric field
•	Migration speed determined by:
o	Magnitude of charge
o	Strength of buffer- >> = more friction
o	Temperature- >> = >>
o	Particle dimensions

Question 7

Haematology

Answer 7

• Counting and separation of RBC, WBC and platelets
• Achieved via coulter counting or flow cytometry
• Coulter counting-
o measures change in resistance in sample as cells pass through it
o provides number and size of cells within sample
• Flow cytometry-
o Cells counted and classified based on light scattering and fluorescent properties

Question 8

Blood gas analysers

Answer 8

• Measure pH, pCO2, pO2, some ions, haemoglobin, bilirubin, lactate, glucose
• pH- electrode measures potential at tip. Resulting voltage proportional to H+
• CO2 combines with H2O to release H+, voltmeter measures H+ but calibrated to read pCO2
• O2 dissolved in electrolyte, voltage applied, current increases, current plateaus when determined by O2 diffusion rather than voltage, plateau correlates to pO2 in electrolytes