The primary objective of instrumentation is to provide the clinician with the best possible data to be of value to the patient.

Thorough understanding of the principles associated with the machines used is quite necessary for the operators in order to have an easier time in performing maintenance procedures, calibration, and in troubleshooting problems that may arise. Automation in the clinical laboratory has been driven by the need to create automated systems which are capable of reducing or eliminating manual tasks in perform analytical procedures, and thus enhancing the reduction of errors.

AUTOMATION
3 stages in automation: Pre-Analytical Stage

The pre-analytical stage is concerned with the handling of the sample or specimen before processing. The two goals involved in this stage are to minimize non-value added steps in the laboratory process and to increase available time for value-added steps in order to produce better results. Automated pre-analytical system attempts to provide the user with some of the tasks necessary to prepare samples for testing, namely: pre-sorting, centrifugation, volume checks, clot detection, decapping, secondary tube labeling, aliquoting, and destination sorting in analyzer racks. Analytical Stage

The analytical stage is primarily concerned with the processing of the sample or specimen. Tasks: 1. Introduction of the sample may be accomplished through the use of peristaltic pumps and positive liquid displacement. 2. Addition of reagent reagent used must be handled, prepared and stored properly. Automated analyzer can be classified based upon reagent. Automated analyzers classified based upon the reagent used: o Open reagent system - a system in which the reagents other than the instrument manufacturers reagent can be used. Close reagent system - the operator can only used the manufacturers reagent.

3. Mixing Mixing devices and techniques used: o o o o o Magnetic stirring Rotating paddles Forceful dispensing Use of ultrasonic energy Use of vigorous lateral displacement

4. Incubation - uses an elongated cuvet path length and fluorocarbon oil incubation bath to maximize result accuracy by enhancing absorbance value, while using microvolume technology for samples and reagent (Bayer Diagnostics) or uses a thermal electric module in the shape of a ring to maintain a constant temperature for analysis (Beckman Coulter). 5. Detection o Absorption spectroscopy automated analyzer. principal means of measurement in

Reflectance photometry adapted to dry slide analysis and used in chemistry laboratories. Fluorescent compounds - used for measurement of drugs, hormones, and vitamins in several immunoassay analyzers.

POST ANALYTICAL STAGE

The post analytical stage plays its role after processing of the sample or specimen. The instrument computer plays a major role in this stage as it represents a means to accomplish several tasks, which include signal processing, data handling and process control. The processing of data by computers has allowed automation of nonisotopic immunoassays, reflectance photometry and other nonlinear assays because computer algorithms can transform nonlinear standard input signal into linear calibration plots. Computers can also perform data correction, subtract blank response, and monitor patients result against reference values.

INSTRUMENTATION

PRINCIPLES USED: SPECTROPHOTOMETRY Spectrophotometry involves the measurement of the amount of light absorbed by a solution and relating it to the concentration of the solution. It uses Beer-Lambert Law (often referred to as the Beers law), which states that the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light, as its fundamental principle. 8 basic components: 1. Light source - light source provides light and must provide enough energy or power to measure the analyte of interest. Tungsten lamp or tungsten-halogen lamp used for wavelengths in the visible region Deuterium lamp used for wavelengths in the ultraviolet region Silicon carbide - used for wavelengths in the infrared region

2. Entrance slit - excludes unwanted or stray light and prevents scattered light from entering the system. 3. Monochromator - produces the light of specific wavelength from the light source. It arranges the wavelength of light in such a way that results to the wavelength required in the process. Quartz prism separates white light into a continuous spectrum and uses the principle of refraction. Diffraction gratings bends light and forms wave front for ultraviolet and near infrared spectrum. It uses the principle of diffraction. Interference filter based on constructive interference of waves, utilizes several mirrors, and uses the principle of reflection.

4. Exit slit - also known as bandpass act as the passage way of the filtered light from the monochromator to the sample cell.

5. Sample holder - also known as cuvet or analytical cell, maybe square or round in shape and is used to hold samples and must be scratch-free so as not to obtain erroneous results. Soft glass cuvet for acidic solutions Borosilicate cuvet for strong basic solution Quartz or plastic cuvet for ultraviolet measurements

6. Photodetector - convert transmitted energy into an equivalent amount of electrical energy. 7. Signal processor - a device that alters the signal and filters it to remove unwanted components. 8. Readout device REFLECTOMETRY Reflectometry makes use of a filter photometer (reflectometer), which measures the quantity of light reflected by a liquid samples that has been dispensed onto a coarse solid support. Reflectometry is used in urine dipstick analysis and dry slide chemical analysis. 2 types of reflectance: Specular reflectance occurs of polished surface Diffuse reflectance occurs on nonpolished surface Monitor Ammeter Galvanometer Recorder

MOLECULAR LUMINESCENCE SPECTROSCOPY (FLUOROMETRY) Fluorometry is based on the principle of luminescence in wherein an exchange of energy occurs when compounds absorb electromagnetic radiation, become excited and return to an energy level lower or equal to their original level. Chemiluminescence is an example. Basic components: Light source

Excitation (primary) monochromator Cuvet Emission (secondary) monochromator Photodetector

NEPHELOMERTY AND TURBIDIMETRY Both nephelometry and turbidimetry are based on the principle of scattering of radiation by particles in suspension. Nephelometry, which is often used in the measurement of antigen-antibody reaction, is the measurement of the light scattered by a particulate solution whereas turbidimetry, commonly used in coagulation analyzers, measurement of antibiotic sensitivities, and quantification of protein concentration in body fluids, is the measurement of the reduction in light transmission caused by particle formation. REFRACTOMETRY Refractometry is based on the principle of light refraction, which is the bending of light. Refractometry is used in measuring protein concentration, specific gravity of urine, and column effluent of high-performance liquid chromatography analysis. OSMOMETRY Osmometry involves the measurement of the osmolality of an aqueous solution and is based on the measuring changes in the colligative properties of solutions owing to variations in particle concentration. FLOW CYTOMETRY Flow cytometry is the measurement of multiple properties of cells suspended in a moving fluid medium. It is used to count and sort cells, is the core component of hematology cell counters, and is the technology used to differentiate white blood cells. ELECTROCHEMISTRY Electrochemistry involves the measurement of the current or voltage generated by the activity of specific ions. Potentiometry the measurement of voltage between two electrodes in a solution Coulometry the measurement of the quantity of electricity needed to convert an analyte to a different oxidation state

Voltametry - a method in which a potential is applied to an electrochemical cell and the resulting current is measure aniodic strippling voltametry - an electrochemical technique used in measuring heavy metals Amperometry - the measurement of the current flow produced by an oxidation-reduction reaction.

CONDUCTANCE Conductance is a measurement of the ability of a solution to carry an electric current. This principle is used monitoring water purity, measuring analytes in blood. It is also the principle used in the components of detectors used in high performance liquid chromatography (HPLC) and gas chromatography (GC), cell counters, and capillary electrophoresis. IMPEDANCE Electrical impedance, a measurement based on the change in electrical resistance across an aperture when a particle in the liquid passes through this aperture, is primarily used in enumerating leukocytes, erythrocytes, and platelets. ELECTROPHORESIS AND DENSITOMETRY Electrophoresis is the separation of charged compounds based on their electrical charge, and in order to obtain a quantitative profile of the separated fractions, the principle of densitometry is used, which is performed on the stained support medium. ISOELECTRIC FOCUSING Isoelectric focusing is a technique which is performed similar to other electrophoresis methods, the difference lies on the medium through which the separating molecules migrate through. In isoelectric focusing, the separating molecules migrate through a pH gradient. This principle is useful in measuring serum acid phosphatase isoenzymes and in detecting oligoclonal immunoglobulin bands in CSF and isoenzymes of creatine kinase and alkaline phosphatase in serum. CHROMATOGRAPHY Chromatography is a method of separation which is based on the different interactions of the specimen compounds with the mobile phase and with the stationary phase as the compounds travel through a support medium. Gas chromatography - useful for volatile compounds 2 types of stationary phases commonly used in gas chromatography:

Gas-solid chromatography- solid absorbent and uses the same material for both the stationary and the support phase Gas-liquid chromatography - uses liquid phases to coat the solid support material

Liquid chromatography - for better separation of thermolabile compounds High performance liquid chromatography - better method of liquid separation over other forms due to a superior resolution, shorter analysis time, and a greatly improves reproducibility

commonly used separation techniques in liquid chromatography: Adsorption (liquid-solid) chromatography - compounds are adsorbed to a solid support

Partition (liquid-liquid) chromatography - separates compounds based on their partition between a liquid mobile phase and a liquid stationary phase coated on a solid support o Normal-phase liquid chromatography - uses a polar liquid stationary phase o Reverse-phase stationary phase liquid chromatography uses a nonpolar liquid

Ion-exchange chromatography - uses column packings that have chargebearing functional groups attached to a polymer matrix and uses the mechanism of the exchange of sample ions and mobile phase ions with the charged group of the stationary phase

Affinity chromatography - uses immobilized biochemical ligands as the stationary phase to separate a few solutes from other unretained solutes

Size-exclusion chromatography - separates molecules according to the difference in their sizes

MASS SPECTROMETRY Mass spectrometry is based on fragmentation and ionization of molecules using a suitable source of energy. The resulting fragment masses and their relative

abundance yield a characteristic mass spectrum of the parent molecule. Mass spectrometry typically involves the following major steps which include, conversion of parent molecule into a stream of ion; separating the ions by mass-to-charge ratio; counting the number of ions of each type or measuring current produced when the ions strike a transducer. SCINTILLATION COUNTER A scintillation counter is an instrument that detects scintillations (flashes of light that occur when gamma rays or charged particles interact with matter) using a photomultiplier tube and counts the electrical impulses produced by the scintillations. Scintillation counting is usually applied in radio immunoassays. 2 types of scintillation method: Crystal scintillation used to detect gamma radiation Liquid scintillation used to count radionuclides that emit beta particles

CAPILLARY ELECTROPHORESIS Capillary electrophoresis is a separation technique that is said to be better than conventional electrophoresis and high performance liquid chromatography due to its short analytical time, resolving power, and microsample volumes. Capillary electrophoresis is applied in the separation of serum proteins and haemoglobin. NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY Nuclear magnetic resonance spectroscopy is a technique for determining the surface of organic compounds. It is non-destructive, but requires a larger volume of the sample compares to mass spectroscopy. Nuclear magnetic resonance is a phenomenon that occurs when the nuclei of certain atoms are immersed in a static magnetic field and exposed to a second oscillating magnetic field. Lipoprotein particle measurement is the most popular application of nuclear magnetic resonance spectroscopy.