Principles of Biomedical
Systems & Devices
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Lecture 17
Clinical Systems
Today Week in PBS&D
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Clinical Laboratory Instrumentation
Spectrophotometry
Autoanalyzers
Chromatology - Gas Chromatgraphs
(Mass) Spectroscopy
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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
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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 Beer’s Law:
Absorbtivity related to the nature of the
absorbing substance and optical wavelength
(known for a standard solution
concentration).
A=aCL
Unknown
concentration
C: Concentration
L: Cuvette path length
Cµ = Cs
Aµ absorbance
As
Standard (known)
concentration
Colorimeter
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Wavelength selector
Focusing lenses
Flame Photometer
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Measures the color intensity of a flame supported by O2 and a specific substance.
Sample’s 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 (50µm), 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.
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Aperture Impedance
(Coulter Counter)
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Vacuum (6"Hg)
Aperture
current
Internal
electrode
100 µm
+
75 µm
External
electrode
-
Blood
cell
suspension
Sample
beaker
Aperture
Aperture tube
Detail of
aperture
(WBC)
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
Diluting
fluid
Diluter I
Lysing
agent
Diluter II
Lysing/
WBC mixing
bath
Hgb
Meter
Triple
C
C
Hgb
C
RBC
bath
C
C
Analyzer computer
Laboratory
DM
Computer system system
Printer
C
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
Color
Nucleated erythrocytescell
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
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
3. Introduces
the sample into
the column
4. The column is where the separation takes place. A glass or metal tube (1 m / ø7 mm) of sufficient
strength to withstand the pressures applied across it. The column contains the stationary phase.
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=
2V
m
×
H2 e
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
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Commercial Autoanalyzers
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