Idea Transcript
Clinical Laboratory Instrumentation
Electrical and Computer Engineering Department
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Clinical Laboratory Instrumentation • Conduct tests on specimen from patients (blood, urine, cerebrospinal fluids) to find information to aid physicians in diagnosis and treatment gy section tests on blood ((Red and white • Hematology blood cells, platelets) using coulter counter • Microbiology tests on tissues and fluids to determine the presence of organisms • Instrumentation is now applied in most if not all applications in the clinical laboratories.
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 C t systems t are usedd
Spectrophotometry • It is based on the face that substances absorb or emit electromagnetic energy at different wavelengths g • Wavelengths used (ultraviolet 200-400 nm), (visible 400 400-700 700 nm), nm) (infra (infra-red red 700 700-800 800 nm) • Most applications use visible light
Principles p and Equations q • Beer's law: • P = Po10-aLC Po : power arriving at the cuvette P : power leaving the cuvette a: absorption coeff. L : Length of the path through the sample C : Concentration of the absorbing sample aLC • % transmittance =%T = P*100/Po =(100)10-aLC • Absorbance: A = log(Po/P) = aLC
Calculation of Concentration • Absorbance of a standard (As) with known concentration Cs of a substance of interest is determined. • Absorbance of the sample (Au) with the unknown concentration Cu is measured. • Beer's B ' llaw iis usedd to t calculate l l t Cu Cu = Cs ((Au/As)
Some Observations on Light • White light contains infinite number of wavelengths l th • The electromagnetic g spectrum p identified as: – Visible (human eye can see colors) from 400 to 700 nm – Ultraviolet from 200 to 400 nm – Infrared I f d ffrom 700 to t 900 nm
Spectrophotometer Block diagram
Power Sources • Hydrogen y g and deuterium discharge g lamps p – Continuous spectrum with most power in IR range – Operating above the nominal voltage increases th power iin visible the i ibl and d UV ranges andd can be b used from 200 to 360 nm
• Tungsten filament – Operation p from 360 to 800 nm
W l Wavelength h Selectors S l • Filters – Glass filters - BW 50 nm – Interference filters - BW 10 to 15 nm
• Monochromators M h t – Prism - BW 0.5 nm • Glass • Quartz, for < 350 nm
– Diffraction grading - BW down to 0.5 nm
Monochromators • Prisms – – – –
Glass Quartz for < 350 nm Spectrum of light produced N li Nonlinear spatial ti l distribution di t ib ti
• Diffraction grating –
More linear spatial distribution
• Bandwidth down to 0.5 nm
Photometric system • Photodetector • Quantum sensors - absorb energy gy of photon p and release electron in a restricted wavelength • photodiode, phototube
–
Photoemissive sensors - photocathode receives photon and emits electron • Phototube, photomultiplier
–
Ph Photoconductive d i cells ll
• Signal processing and display – – –
Amplifiers Linearizers Signal processing devices, integrators, differentiators, filters – Analog and digital display – Recording R di devices d i
Photomultiplier oo u p e
An incoming photon strikes the photocathode and liberates an electron. This electron is accelerated toward the first dynode, which is 100 V more positive than the cathode. The impact liberates several electrons by secondary emission. They are accelerated toward the second dynode, which is 100 V more positive than the first dynode, This electron multiplication continues until it reaches the anode, where currents of about 1 mA flow through RL.
Flame Photometers • Differs from spectrophotometers in three ways: – Power source and sample holder functions are combined in the flame – Objective j is measurement of sample's p emission rather than absorption of light – Can determine only concentrations of pure metals
Types yp of Flame Photometers
Atomic At i emission i i • Sample and reagent and combined. A nebulizer applies p and the solvent to the flame. The solvent evaporates yields atoms that emit light. • Only about 1 % goes from ground state to excited state • Used for Na+, K+ and Li+ • Li+ does not exist in the body normally and it is used as a reference
Types yp of Flame Photometers
Atomic At i absorption b ti • Atoms in flames absorb energy at characteristic wavelength. g , • Power source emits ppower at characteristic wavelength, atoms gets excited and emits light that is detected. • Energy is absorbed as the atom goes from ground state to excited state • Used for calcium, lead, copper, zinc, iron and magnesium
Block diagram of a fluorometer
Automated Chemical Analyzer • Utilize spectrophotometric methods for g the actual measurement of interest. making • Two examples –B Beckman k Synchron S h CX4 – Dupont Automatic Clinical Analyzer (ACA) – Both equipments differ in how specimen is used, diluted,, and computation p
• Check the text book for detailed description Electrical and Computer Engineering Department
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Chromatology gy
• • • • • •
Separation of mixture of substances into component parts. Differences in the rate of movements of particles are used to separate these components from each other Patient sample (with a volatile solvent) flash evaporates as it enters It is carried out through a column (1 m long and about 7 mm in diameter) packed with solid material that causes separation of material An electric field is applied after the column and the generated current is proportional to the quantity present in the column Different components arrive at the detector at different times
Hematology • Blood: –
Formed elements • Red blood cells (RBC): 4.6 to 6.2*106/µl in men and 4.2 to 5.4*10 5.4 106/µl in women • White blood cells (WBC): 4500 to 11000/µl • Platelets: 150,000 to 400,000/µl • Water
• Hematocrit (HCT) –
Expressed p in the ratio of formed elements to total volume – 40 to 54 % in adult men – 35 to t 47 % in i adult d lt women
• Hemoglobin (Hb) in grams/dl – –
13.55 to 18 g/dl in adult men 13 12 to 16 g/dl in adult women
Electronic measuring techniques • Two major types utilizing two concepts: 1. Changes g in the electrical resistance of a solution when a formed blood element passes through an aperture. p ((Coulter corporation, p , and Baker Diagnostics) 2 Deflections of a light beam caused by the 2. passage of formed blood elements to make the measurements (Technicon corporation) measurements.
Electrical and Computer Engineering Department
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Coulter Counter • • • •
•
Blood elements are separated through a centrifuge system Formed elements are diluted with a substance having chemical characteristics as plasma p Lyzing solution ruptures RBC to release hemoglobin in the solution and spectrophotometry is used to find concentration A pump ddraws the h fl fluid id through h h the h aperture. Two electrodes l d across the h aperture pass current through the solution, as each WBC passes through the aperture, the volume of the solution is reduced. The resistance of the WBC is >>> fluid electric pulse is generated with an amplitude proportional to WBC volume The same technique is used to calculate RBC count
Electrical and Computer Engineering Department
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A block diagram of a Coulter Model STKS Dilutingg fluid
Dil Diluter I
• Blood elements are p separated
WBC stabilizing agent
Lysing agent
Diluter II
Lysing and mixing
Lysing and mixing
Triple transducer WBC bath module
Hgb
Hemoglobinometer
RBC bath C
C
C
C
C
Analyzer computer
Laboratory computer system
Data management system
Printer
C
Coulter STKS aperture p bath Vacuum (6"Hg)
Aperture current
Internal electrode
100 m +
75 m
External electrode -
Blood cell suspension
Sample beaker
Aperture
Aperture tube
Detail of aperture (WBC)