MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

1. MLAB 2401: Clinical ChemistryKeri Brophy-Martinez Designs in Automation Part Two

2. Osmometry • Two instrument types • Freezing point depression • Vapor pressure (dew point) • Clinical Applications • osmolality • Osmometry • Serum and urine • Principle • Measure osmolality indirectly by measuring a colligative property paralleling osmotic pressure.

3. Osmometer / Osmometry Vapor Pressure Depression Analyzers • Sample is sealed within a small chamber • Humidity equalizes between the air in the chamber and the specimen • Electrical potential is passed through a wire in the chamber by a thermocouple which measures the temperature at which the chamber is saturated with vapor from the specimen • The thermocouple also transfers heat from the chamber and cools it below the dew point • As the current from the thermocouple is turned off, the temperature in the chamber rises and vapor condenses on the wire • A temperature equilibrium occurs when evaporation equals condensation on the wire , affecting its voltage • The vapor pressure is directly proportional to the thermocouple voltage

4. Osmometer

5. Fluorometry • Clinical Applications • protoporphyrin, some therapeutic drugs, a few coag applications • Advantages of fluorescence: • Very specific and sensitive • Disadvantages of fluorescence: • Few molecules fluoresce • Very susceptible to pH and temperature changes (Quenching) • limitations of detection, etc.

6. Fluorometer: Principle 1. Fluorometers measure substances that absorb short λ and release energy of longer wavelength; for those few molecules are capable of absorbing light of one wavelength, then emitting light at a different, longer wavelength. 2. Mercury arc or xenon arc lamp produces short λ (UV) light which is passed through a monochromator. 3. Monochromatic light of an appropriate short λ passes through a quartz / fused silicon cuvet holding the specimen. 4. The light energy is absorbed by the molecules which then release some of the energy as a longer wavelength. 5. A second monochromator at a 900 angle to the light source filters out λ other than the long wavelength being emitted. 6. The amount of light being released is proportional to the fluorescing molecules.

7. Fluorometry

8. Fluorometry • The detector is located at a 90° angle from the initial light source • This eliminates any interference from the initial light source and detects only fluorescent light.

9. Scintillation Counters • Clinical Applications • Trace levels of hormones and drugs • Principle • A particle tagged with a radio nuclide emits gamma rays which strike a detector of a scintillation counter producing an electrical pulse of a size proportional to the energy of the gamma ray striking it. • Gamma rays produced as a result of an unstable nucleus shifting to become more stable. - compared to light, which is excited electrons returning to ground state.

10. Scintillation counter • Basic instrument and principle • The tube with gamma emitting substance (I125) is placed into the well in the center of the detector. The detector contains a crystalline substance, usually NaI, which is a phosphor

11. Scintillation counter • Gamma rays striking the phosphor cause it to release a flash of visible or U.V. light (scintillation) - it gives up visible light when struck by gamma energy • These flashes are then picked up and amplified by the photomultiplier component of the detector • The greater the gamma energy, the brighter the flash

12. Scintillation counter • Calculations - to determine the concentration of unknown, compare the unk’s count to the std’s count. • Unknown’s Concentration = Unknown’s count / Standard’s count X Standard’s concentration