Turbidimetry
Turbidimetry is the measurement of the reduction in light transmission caused by particle formation. Light transmitted in the forward direction is detected. The amount of light absorbed by a suspension of particles depends on the specimen concentration and on the particle size. Solutions requiring quantitation by turbidimetry are measured using visible photometers or visible spectrophotometers. High sensitivity has been achieved through photodetectors that can quantify small changes in signals. Sensitivity comparable to nephelometry can be attained using low wavelengths and high-quality spectrophotometers.
Many clinical applications exist for turbidimetry. Various microbiology analyzers measure turbidity of samples to detect bacterial growth in broth cultures. Turbidimetry is routinely used to measure the antibiotic sensitivity from such cultures. In coagulation analyzers, turKdimetric measurements detect clot formation in the sample cuvettes. Turbidimetric assays have long been available in clinical chemistry to quantitate protein concentration in biological fluids, such as urine and cerebrospinal fluid.
Refractometry is based on light refraction. When light passes from one medium into another, the light beam changes its direction at the boundary surface if its speed in the second medium is different from that in the first. The ability of a substance to bend light is called refractivity. The refractivity of a liquid depends on the wavelength of the incident light, the temperature, the nature of the liquid medium, and the concentration of the solute dissolved in the medium. If the first three factors are held constant, the refractivity of a solution is an indirect measurement of the solute concentration. Refractometry has been applied to various measurements, for example, total serum protein concentration, specific gravity of urine, and column effluent of high-performance liquid chromatography analysis.
Osmometry
Osmometry is the measurement of the osmolality of an aqueous solution such as serum, plasma, or urine. As osmotically active particles are added to a solution causing its osmolality to increase, four other properties of the solution are also affected. These properties are osmotic pressure, boiling point, freezing point, and vapor pressure. They are called colligative properties of the solution because they can be related to each other and to the osmolality. As the osmolality of a solution increases, first the osmotic pressure increases, second the boiling point is elevated, third the freezing point is depressed, and fourth the vapor pressure is depressed. Osmometry is based on measuring changes in the colligative properties of solutions that occur owing to variations in particle concentration. Freezing-point depression osmometry is the most commonly used method for measuring the changes in colligative properties of a solution. Therefore, only components of a freezing-point osmometer are described in detail. A freezing-point osmometer consists of a sample chamber containing a stirrer and a thermistor temperature-sensing device connected to a readout device. The sample is rapidly supercooled to several degrees below its freezing point in a refrigeration chamber containing antifreeze. The sample is then agitated with the stirrer to initiate freezing. As the ice crystals form, heat of fusion is released from the solution. The rate of heat of fusion released from the ice being formed rapidly reaches equilibrium with the rate of heat removed by the colder temperature of the sample chamber. This equilibrium temperature, known as the freezing point of the solution, stays constant for several minutes once it is reached. This freezing point is detected by the thermistor, and the osmolality of the sample is displayed in units of milliosmoles per kilogram of water.
Freezing-point osmometers in common use include the Micro Osmette and Osmette II models. The Micro Osmette model measures samples of 50 uL in volume, and the Osmette II measures samples of 200 uL in volume. Both models have a measurement time of 180 seconds.
Flow Cytometry
Flow cytometry measures the properties of cells suspended in a moving fluid medium. All cells pass single file through a sensing point, where they are intercepted typically by either water or air cooled argon laser beam. The transmitted light consists of both scattered light forward and 90 degree and fluorescent light. The light is directed by lenses and focused onto appropriate PMT. An analogue signal from the PMT is converted to a digital signal that the computer can use for quantitation.
