Clinical Laboratory Instrumentation

• Responsible for analyzing patient

specimens to provide information
– to aid in diagnosis of disease
– to evaluate effectiveness of therapy
• Major sections:
– Chemistry lab (Blood, Urine, fluids)
– Hematology (Elements of blood)
– Microbiology (Tissues to search for organisms)
– Blood bank
 Operations in a Clinical
Laboratory
• Sample handling
• Performing tests
• Discards used samples safely
• Information management
– Analyzes and reports results
– Stores results in a data base
– Computer systems are used
 Spectrophotometry
• Basis of many devices in Clinical Lab
• Easy, accurate, precise, and stable
• Concept:
– Substances absorb or emit electromagnetic
energy at different wavelengths. Ultraviolet,
visible and near Infrared.
• Two types of Spectrophotometer:
– Photometer, no filter
– Colorimeter, glass filter that selects λ range
 Some Observations on Light
• Photon is a light particle with zero resting mass
• Color perception is measured by special cells
• Colors are classified as pure and non-pure colors
• White light contains infinite number of λ
• The electromagnetic spectrum identified as:
– Visible (human eye can see colors) from 400 to 700
nm. Mostly used in spectrophotometry.
– Ultraviolet from 200 to 400 nm
– Infrared from 700 to 900 nm
Block Diagram-Spectrophotometer

• Apply light
• Select λ (i.e. select element you are searching for) using
a narrow-band light filter – mono-chromator
• Apply selected light to sample
• Measure returned energy
concentration of element
• If the element of interest is not light sensitive, add
reagent to react with element of interest
 Power Sources
• Hydrogen and deuterium discharge lamps
– Continuous spectrum with most power in IR range
– can be used from 200 to 360 nm through higher V.
• Tungsten filament
– Operation from 360 to 800 nm
– Problems due to aging and voltage dependence
– self calibration is possible
• Power sources used:
– Batteries, mainly Ni-Cd rechargeable, limited use
– Constant voltage transformers
– Electronic power supplies
 Light Sources

(green) Tungsten
(red)

• Lasers (monochromatic):
• Tungsten lamps
– Argon 515 nm
• Arc discharges – He-Ne 633 nm
• Light emitting diodes – Ruby 693 nm
(LED's) – Nd:YAG 1064 nm
– CO2: 10,600 nm (not shown)
 Optical Filters

Kodak
Corning

• Glass and gelatin filters

• Germanium lenses for long λ
• Two Polaroid filters
• Interference filters
• Diffraction grating
 Wavelength Selectors
1. Filters
a. Glass filters - BW 50 nm
b. Interference filters - BW 10 to 15 nm
• require glass filter to eliminate harmonics
2. Monochromators
– Prism - BW 0.5 nm
• Glass
• Quartz, for λ< 350 nm
– Diffraction grating - BW down to 0.5 nm
 1-a Glass Filters
• Glass is treated with some materials (paint)
to absorb some wavelength and transmit
some others
• Multi-layer of glass filters are used to obtain
high-pass, low-pass and band-pass
characteristics
• Bandwidth around 50 nm, hence can't
distinguish between particle whose optical
spectrums are close to each other
 1-b Interference Filters
• Reflected surfaces are spaced at a distance
causing light to go back and forth
• Distance equals to desired wavelength

desired wavelength is enhanced since it is
in-phase, while un-desired light is cancelled
since it is out of phase.
• Harmonics can also pass
must eliminate
using glass filters
• Interference filters can distinguish between
elements with close wavelengths.
 2-Monochromators
• Prisms
– Glass
– Quartz for λ< 350 nm
– Spectrum of light produced
– Nonlinear spatial distribution
• Diffraction grating
– More lin. spatial distribution
– Stray light due to impurities
in grating
– Harmonics
• Bandwidth down to 0.5 nm
 Cuvette
• Holds the sample
• Optical Characteristics must not alter the
spectral characteristics of light
entering/leaving the cuvette
 Sample
• Substance resulting from interaction of the patient
specimen and appropriate reagent
• Substance absorbs light according to Beer’s law
• Beer's law: Pt = Po10-aLC where
• Po : radiant energy arriving at the cuvette
• Pt : Radiant energy leaving the cuvette
• a: absorptivity of the sample (extinction coefficient)
• L : Length of the path through the sample
• C : Concentration of the absorbing sample
• % transmittance (T) = Pt*100/Po =10-aLC
• Absorbance
• A= log (Po/Pt) = log(100/%T) = 2 - log(%T) = 2 - (2 - aLC);
• ∴ A = aLC
 Calculation of Concentration
• Absorbance in standard (As) (standard
reagent with know concentration Cs) is
determined.
• Absorbance of the sample (unknown) (Au)
is measured.
• Using the Beer's law:
Cu = Cs (Au/As)
where Cu and Cs are concentrations of unknown
and standard in the sample respectively
Beer's law: T = Pt*100/Po =10-aLC
Pt = Po10-aLC
Absorbance:
A = aLC

Principles
 Detectors-Photometers
• Photodetector
– Quantum sensors
– Photo-emissive sensors
– Photo-conductive cells
– Photo-junction sensors
– Photo-voltaic sensors -
solar cells
• Signal processing and
display
– Amplifiers
– Linearizers
– Signal processing
devices, integrators,
differentiators, filters
– Analog and digital
display
– Recording devices
Diagram of an Optical Instrument

Schematic diagram for highest efficiency

Solid-state lamps and detectors may simplify the system

 Geometrical Optics
• Lamps • Filters
• Lenses • Scattered radiation
– collimating the beam – flat black painting
– focusing the beam – stops
• Mirrors
– full mirrors
– half-silvered mirrors
– curved mirrors
Fiber Optics

n1sinθ1 = n2sinθ2
Radiation Sensors (Photo-detectors)

• Thermal sensors • Photo-conductive cells

• Quantum sensors • Photo-junction sensors
• Photo-emissive • Photo-voltaic cells
sensors
The Photomultiplier
 Photo-Junction
Sensors

Photodiode, phototransistor,
photo darlington transistor,
photo-unijunction transistor,
photon (opto) couplers
Spectral Response Including
Source, Filter and Detector
Simplified Schematic
Diagram of a
Spectrophotometer
 Utilization of Reagent
Unknown Reagent
Unknown Color Light source Light
chemical
chemical density
concentration
concentration intensity
Electrical Photodetector
Reading of
meter Electrical
unknown
concentration signal

• Most substances do not have energy absorption

characteristics. Special chemicals are used.
• Reagent (special chemicals) must have:
– Complete reaction with the unknown
– Reaction product should have strong color
– Unknown should not have color itself
Clinical Lab-Flame Photometers
Flame Photometer- Atomic Emission
• Differ from spectro-photometer
– Power source and Sample are combined
– Measures emission of light not absorption
– Can determine concentration of only metals (Na+, K+)
• Sample
nebulizer (converts liquid to gas)

inject to flame
evaporation
particles of
substance with high energy remain
particles
release energy at certain wavelength
lens and
photo-detector measures concentration.
Flame Photometer- Atomic Absorption
• Used with lead, cupper and calcium
• Concept:
1. Use a power source (light) with filament made
of element to be detected
2. Keep the cathode of lamp in vacuum
3. Heat the lamp
release atoms from cathode
4. Cathode atoms will collide with sample atoms

energy release proportional to
concentration of atomes in sample
 Types of Flame
Atomic emission Photometers

Atomic absorption
 Automated Chemical Analyzers
• Increase productivity and decrease response
time for emergency request (STAT request)
• Utilize spectrophotometric methods
• Three important devices:
– Technicon SMA 12/60 & SMAC
– Beckman Synchron CX4
– DuPont Automatic Clinical Analyzer (ACA)
Continuous Flow Analyzer
Autoanalyzer
 CX4 measurement
read window

Synchron CX4 measurement read window for a rate-type measurement.

Block Diagram
of ACA
• Sample kit holds the patient
samples with ID attached.
• ATP’s needed for each
sample loaded behind the
sample kit on conveyor.
• The last ATP followed by a
special end-of-run kit.
• At filling station, sample
mixed with diluent (may be
different for each determin.)
and combined solution is
injected to each ATP.
• ATP to preheater, sample kit
to sample-kit exit tray.
 Hematology
• Blood is composed of:
– Formed elements, RBC, WBC, Platelets
– Plasma
• Substances in solution
• Water
• Hematology counts # and determines
characteristics of formed elements in blood
– Packed RBC volume = hematocrit (HCT)
– Hemoglobin (Hb) in grams/dl
Blood Spun in a Centrifuge
Hematocrit
Determination
 RBC Volume and Hb Concent.
• Mean corpuscular volume - MCV: 82 to 98 µm3
• Mean corp. hemoglobin - MCH : 27 to 31 pg
• Mean corpuscular hemoglobin content -MCHC:
32 to 36 %
• MCV = 10 HCT / RBC count
• MCH = 10 Hg / RBC count
• MCHC = 100 Hg / HCT
• RDW - volume distribution width for RBC
 Chromatology
• Separating a mixture of substances into components parts
Differences in the rate of movement of components of a mixture
while in mobile phase are used to separate components
Can determine drugs taken in overdoses
 Gas Chromatology
How it works:
• Injector introduces sample to pressurized gas that carries the sample
through the 1 meter long 7 mm diameter column
• Column is coated with solid supports with a small size to produce the
separation of substances.
• Temperature is increased along the column to ensure maximal separation
efficiency
• A detector at the end of the column provides an electrical output
proportional to the quantity of the compound.
• Recorder with x axis for time and y axis to amplitude records detector
output
 Electrophoresis
• Measure quantities of proteins in plasma ,
Cerebral Spinal fluids (SPF) and urine
• It is defined as the movement of solid phase with
respect to a liquid (buffer)
• Buffer is a solution to carry current and maintain
PH.
• How it works?
– Sample applied to the buffer
– Due to electric field applied, samples with same size,
charge and shape migrate at the same rate resulting in
separation of particles into zones.
– Densitometer is used to measure the concentration of
each protein on the strip.
Electrophoresis

Cellulose acetate electrophoresis

 Hematology
• Blood:
– RBC
carry oxygen (4.6-6.2x106/µl)
– WBC
defense (4500-11000 /µl)
– Platelets
coagulation (150,000-400,000 µl)
– Plasma
liquid that carries everything
• RBC Volume and O2 concentration
Electronic Devices for Measuring
Blood Characteristics
• Dark field
– used in Technicon instrument
– deflection of light beam caused by the passage
of formed elements.
• Electrical resistance
– principle used in Coulter counter
– resistance of a solution as formed elements pass
through an aperture
– mostly used clinical method.
 Coulter Counter
• How it works:
– Add anti-coagulant chemical to blood (why?)
– Dilute specimen with a solution similar to
plasma
– Split diluted specimen to two parts
• Mixing and lyzing chamber
Measure Hb
and WBC count
• Diluter II
Measure RBC count
– Increase accuracy by using 3 counters in the
counting bath
 Hemoglobin (Hb) Concentration
• Objective is to prepare specimen for measuring
Hemoglobin (Hb) and WBC
– Lyzing agent breaks the RBC to release the Hb into
solutions. WBC are not affected with this agent.
– Specimen passes through WBC bath and used as a
cuvette is to measure Hb concentration
 WBC counts
• How it works?
• Vacuum pump draws specimen out of
WBC bath through the aperture
• Current passes from the electrode in
bath to the electrode in the tube
through the aperture.
• As each WBC/RBC passes through the
aperture, a voltage pulse is generated in
the circuit proportional to the volume
of cell.
• A threshold related to the volume of
the cell is used to determine the
passage of a cell.
• Pulses enter to an integrator circuit that
produce a dc voltage proportional to
the cell count
Coulter STKS Aperture Bath
Accuracy and Correction of counts
• 3 counters are used, and the results are averaged if readings are
close to each other.
• If one reading if off shoot, then two are used for average.
• If all three are away from each other beyond a certain limit, the
counting is repeated.
• Correction for coincide (two cells passing together) is done
through statistical analysis.
Diluter II in the Coulter
Model STKS Counter
 RBC Count (Diluter II)
• Same counting technique
• Cells with volume greater than 35fl are
classified as RBC
• Cells with volume in the 2-20 fl range are
classified as platelets.
• Histogram with detected volumes is
generated to yield counts.
The Counting Process
 WBC Differential Count
• Coulter measures the count for the five basic white blood
cell types, each WBC cell type has its' own unique features
– Neutrophils
– Eosinophils
– Basophils
– Lymphocytes
– Monocytes
• WBC differential bath is used
– RBC are removed by lyzing, and WBC are further stablized
– Flow cytometry is used to measure counts for each element.
 Flow Cytometry
• Light beam is presented to a stream of fluid
• Detectors detect the scattered and reflected
light of the fluid
• Different elements reflect differently.
• Use the reflected light to measure the
volume of each element of WBC