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• Johnree A. Evangelista

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• Absorbancia
• Leucocito
• Espectrofotometria
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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)

• Tungsten lamps • Arc discharges • Light emitting diodes (LED's)

• Lasers (monochromatic): – – – – –

Argon 515 nm He-Ne 633 nm Ruby 693 nm Nd:YAG 1064 nm CO2: 10,600 nm (not shown)

Optical Filters

Corning

• • • • •

Kodak

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: Pt = Po10-aLC

T = Pt*100/Po =10-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 • Lenses – collimating the beam – focusing the beam

• Mirrors – full mirrors – half-silvered mirrors – curved mirrors

• Filters • Scattered radiation – flat black painting – stops

Fiber Optics

n1sinθ1 = n2sinθ2

Radiation Sensors (Photo-detectors)

• Thermal sensors • Quantum sensors • Photo-emissive sensors

• Photo-conductive cells • Photo-junction sensors • Photo-voltaic cells

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 Unknown chemical chemical concentration concentration

Reagent

Reading of unknown concentration

Color Light source Light density intensity Electrical Photodetector meter Electrical 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

Atomic emission

Atomic absorption

Types of Flame Photometers

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