PBS&D Fall 2004 Polikar

Principles of Biomedical Systems & Devices

Lecture 17 Clinical Systems

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Today Week in PBS&D

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Clinical Laboratory Instrumentation

Spectrophotometry Autoanalyzers Chromatology - Gas Chromatgraphs (Mass) Spectroscopy

Havent had a quiz for some time

Clinical Laboratory Instrumentation

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Clinical Laboratory: Analyze patient specimens for diagnostic and therapy efficiency measurements
Chemistry section: Blood, urine, CSF, etc. determine how much of a clinically important substance is present Hematology section: Number and characteristics of formed elements in blood (RBC, WBC, platelets, etc.), blood clotting tests Microbiology / Hematology section: test blood and tissue cultures for presence of microorganisms Blood bank section: Determination of blood type

Clinical instruments must be: exceptionally accurate and precise, with fast response!

Clinical Instrumentation
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Typical devices used in the clinical laboratory

Colorimeter, an optical device to measure color concentration of a substance in a solution following the reaction between the substance and a reagent. Flame photometer, an optical device to measure the color intensity of substances, such as sodium, potassium that have been aspirated into a flame Spectrophotometer, also an optical device that measures light absorption at various wavelengths given a liquid sample (also used to refer the above two in general) Blood cell analyzer, measures RBC and WBC count per volume (several methods) pH and blood gas analyzer measure blood acidity, Po2, Pco2 Chromatograph, an electromechanical device to separate, identify and measure the concentrations of substances in a liquid medium Autoanalyzer, an electromechanical electronic device that sequentially measures and displays blood chemical analysis, by mixing several reagents with the blood (automated colorimeter)

Colorimeter
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Measures the color concentration of a substance in a solution by detecting the color light intensity passing through a sample containing the substance and a reagent Optical color filters are used to detect the color wavelength of interest. E.g., urine passes yellow light and absorbs blue and green Laser LEDs are preferred if their wavelength is suitable due to purity of the monochromatic color.

Colorimeter
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Transmittance

I1 T= 100% I0 I 2 = TI1 = T 2 I 0

Absorbance

I0 1 A = log = log I1 T

IF the path length or concentration increases, the transmittance decreases and absorbance Unknown / known increases, a phenomenon expressed by Beers Law:
Absorbtivity related to the nature of the absorbing substance and optical wavelength (known for a standard solution concentration).

A=aCL

Unknown concentration

C = Cs

A absorbance As

C: Concentration L: Cuvette path length

Standard (known) concentration

Colorimeter
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Focusing lenses

Wavelength selector

Flame Photometer
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Measures the color intensity of a flame supported by O2 and a specific substance. Samples emission of light is measured (rather than the absorbance of light). Typically used to determine the conc. of pure metals and/or Na+, K+, Li+ and Ca++

Spectrophotometer
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The general name given to the group of instruments whose principle of operation is based on the fact that substances of clinical interest selectively absorb or emit EM energy (light) at different wavelengths. Depending on the substance being measured, the wavelength used is typically in the ultraviolet (200-400 nm), visible (400-700nm) or infrared (700 to 800 nm) range. Spectrophotometer can be used to determine the entity of an unknown substance, or the concentration of a number of known substances. The type of source / filters used typically determines the type of the spectrophotometer.

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S1: Disperse the light S2: Select the desired wavelength

Rays of light bend around sharp corners, where the amount of bending depends on the wavelength! This results in separation of light into a spectrum at each line

Diffraction Spectrophotometer

Spectrophotometers
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Blood Measurements
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Two main techniques for blood cell count

Aperture impedance method: When blood is mixed with a diluting solution, the resistivity of the blood cells are higher than those of the surrounding fluid. The sensor cell consists of two cambers, separated by a small aperture (50m), and a pair of electrodes is placed one on each side of the chamber, measuring the resistance of the path through the aperture. Flow cytometry cell counters: cells are passed through a single cell wide chamber where they are illuminated by laser; from the scattered light, one can count the each cell passing by.

Aperture Impedance (Coulter Counter)

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Resistance is low when there is no blood cell passing, and high when a blood cell does pass.

Aperture Impedance (Coulter Counter)

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Vacuum (6"Hg) Aperture current Internal electrode

100 m +

External electrode
-

75 m

Sample beaker

Blood cell suspension

Detail of aperture (WBC)

Aperture

Aperture tube

Aperture Impedance (Coulter Counter)

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Aperture Impedance (Coulter Counter)

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How are different cell types differentiated from each other? WBC stabilizing
Various lysing / mixing agents are added to the blood that selectively ruptures or transforms the cells so that they can be separated from each other either by volume or by pattern recognition techniques
agent Lysing mixing Triple

Diluter I Lysing agent Lysing/ WBC mixing bath Hgb Meter Hgb

Diluting fluid Diluter II

RBC bath

Analyzer computer Laboratory DM Computer system system

Printer

Pattern Recognition for blood cell count

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Video scanner
Stage and focus motor drives

Color analyzer

Image memory

Reference memory

Keyboard

Cytoplasm Neutrophils morphology Bands Normal Nucleus morph. Eosinophils cell Granularity Basophils counters Pattern Monocytes Nucl./cytop.ratio Morphological Recognition Lymphocytes analyzer Chromatin pattern "Suspect Algorithm Atypical lymphocytes Blast-like cells cell Color Nucleated erythrocytes counters Other immature cells Vacuolization Video display Ticket printer

Flow Cytometry
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The blood is actually split into different chambers, where in each chamber it is diluted / mixed to differentiate different cell types. WBC and RBC are separated (using lysing)

Chromatography
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A group of measurements for separating a mixture of substances into components parts The chromatograph utilizes an adsorptive medium, which when placed in contact with a sample, adsorbs the various constituents of the sample at different rates. In this manner, the components of a mixture are separated.
Discovered by the Italian-born Russian botanist Mikhail Tswett. Tswett separated plant pigments (chlorophylls) by pouring petroleum-ether extract of green leaves over a column of powdered calcium carbonate in a vertical glass tube. As the solution percolated through the column the individual components of the mixture migrated downward at different rates of speed, so that the column became marked with horizontal bands of colors, called a chromatogram. Each band corresponded to a different pigment. Today, the name chromatography is a misnomer, since the rate at which the substance percolates, not the color, is used to separate the compounds.
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/chroanim.html

Chromatography
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A chromatograph consists of a mobile phase, comprised of a solvent into which the sample is injected the solvent and sample flow through the column together - and stationary phase where the material in the column for which the components to be separated have varying affinities. The materials which comprise the mobile and stationary phases vary depending on the general type of chromatographic process being performed.
Gas Chromatography: The mobile phase in gas chromatography is generally an inert gas. The stationary phase is generally an adsorbent or liquid distributed over the surface of a porous, inert support. Liquid Chromatography: The mobile phase in liquid chromatography is a liquid of low viscosity which flows through the stationary phase bed. This bed may be comprised of an immiscible liquid coated onto a porous support, a thin film of liquid phase bonded to the surface of a sorbent, or a sorbent of controlled pore size.

Chromatography
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1. N2 or He, carries and sweeps the sample and the solvent in which it travels through the separation 5. After the sample is flushed or displaced from the stationary chamber (the column), this constitutes phase, the different components will elute from the column at the mobile phase of the measurement different times. The components with the least affinity for the stationary phase (the most weakly adsorbed) will elute first, while those with the greatest affinity for the stationary phase (the most strongly adsorbed) will elute last. 2. Temp / pressure / pH are controlled in a particular sequence for maximal efficiency of separation 3. Introduces the sample into the column 4. The column is where the separation takes place. A glass or metal tube (1 m / 7 mm) of sufficient strength to withstand the pressures applied across it. The column contains the stationary phase. 6. A detector analyzes the emerging stream by measuring a property which is related to concentration and characteristic of chemical composition. For example, the refractive index or ultra-violet absorbance is measured

Chromatography
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A continuous flow of solvent carries a solution of solutes A and B down a column. (a) As the solvent carries the two solutes down the column, separation of the solution begins. (b) Later in time, solute B starts moving faster rate than A. (c) In (d), solute B emerges first, while solute A finally emerges in (e). Solute A has a greater affinity for the stationary phase than solute B. By varying the pH of the solvent or temperature of the column, the output of the column can be significantly altered, such as the timing of when individual species emerge. http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/chromintro.html

The Chromatogram
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Since the sample is separated in the column, different peaks on the chromatogram correspond to different components in the sample mixture. The chromatograms show the results of separations of protein mixtures by ion exchange chromatography. The lettered peaks correspond to different proteins (A = ovalbumin, B = conalbumin, C = cytochrome c, D = lysozyme). The separation corresponding to the chromatogram on the left was performed at pH 5.85, while the one on the right was performed at pH 6.5. It is evident that operation conditions such as pH and temperature have a significant effect on the output.

Chromatography
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Various types of chromatography are available, each differing with the mobile / stationary phase, or the type of the column / detector\
Liquid chromatography Gas chromatography Adsorption chromatography Ion exchange chromatography Partition chromatography Molecular exclusion Affinity chromatography
See http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/chromtypes.html for more information

Chromatography
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Mass Spectroscopy
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A mass spectrometer produces a stream of charged particles (ions) for the substance(s) being analyzed, separates these ions into a spectrum according to their mass-to-charge ratios, and then determines the relative quantity of each ion present in the substance.
Gas mixture is drawn into an ionizing chamber kept at very low pressure (10-6 torr) The gas molecules to be measured are bombarded by a stream of high velocity e- to produce (+) ions, which are then accelerated into a dispersion chamber in a beam form The ion beam is then sorted into its components on a molecular mass basis The high velocity ion beam is subjected to a high magnetic field H, which causes the ions to deflect and follow a curved trajectory under the field. The curve is least for heaviest ions. By strategic location of an electrode, individual species of ions can be collected. The radius of the trajectory depends on the speed (accelerating voltage), mass, charge and the strength of the magnetic field:

r=

m H2 e

2V

Mass-to-charge ratio

Mass Spectrometer
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Mass Spectrometer
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Dispersion techniques can be varied

Multiple electrodes can be used to collect the ion currents produced by each ion species, strategically placed at the locations the individual ions are expected to fall based on their m/e ratios A fixed electrode can be used along with varied accelerating voltage; ions of different masses can then be collected sequentially using the single electrode A single electrode itself can be scanned over the possible landing locations of the ions

The ion current measured by the collector is proportional to the partial pressure of the sample substance in the gas mixture.

Advantages / Disadvantages
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The MS can identify a single individual gas species in a complex gas mixture. Incredibly small amounts of gas would be adequate, However; MS is unable to distinguish different gases with the same molecular weight, e.g., CO (12+16) and N2 (14*2) cannot be distinguished from each other, nor O2 and CO2 in the presence of N2O. For a demo of MS see http://www.colby.edu/chemistry/OChem/DEMOS/MassSpec.html

Autoanalyzers
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An autoanalyzer sequentially measures blood chemistry through a series of steps of mixing, reagent reaction and colorimetric measurements. It consists of
Sampler: Aspirates samples, standards, wash solutions into the system Proportioning pump: Mixes samples with the reagents so that proper chemical color reactions can take place, which are then read by the colorimeter Dialyzer: separates interfacing substances from the sample by permitting selective passage of sample components through a semi permeable membrane Heating bath: Controls temperature (typically at 37 C), as temp is critical in color development Colorimeter: monitors the changes in optical density of the fluid stream flowing through a tubular flow cell. Color intensities proportional to the substance concentrations are converted to equivalent electrical voltages. Recorder: Displays the output information in a graphical form.

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Autoanalyzers

Commercial Autoanalyzers
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