BIO-CHEMISTRY ANALYZER

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BIO-CHEMISTRY ANALYZER
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DIRECTORATE OF BIOMEDICAL ENGINEERING
PREPARED PRESENTED BY
Eng . Khaled Masalha
Eng . Anton Khleif
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Contents

• Principals of Spectrum
• Beers Law
• Block diagram
• Sample reading
• Chemistry block diagram
• Measurement Principles of Chemistry analyzer
• Photometry Analyzer
• Photometric Measurements
• Kinetic or Enzymatic Measurements
• End Point Procedures
• Application
• Units
• BASIC ISE
• ISE Theory
• ANALYTICAL MEHOTDS
• SAFETY REGULATIONS
• Trouble shooting

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Principals of Spectrum

• Many compounds absorb ultraviolet (UV) or visible
  (Vis.) light. The diagram below shows a beam of
  monochromatic radiation of radiant power P0,
  directed at a sample solution. Absorption takes
  place and the beam of radiation leaving the
  sample has radiant power P.

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Beers Law

• The amount of radiation absorbed may be measured
  in a number of ways Transmittance,
• T P / P0 Transmittance,
• T 100 T Absorbance,
• A  log10 P0 / PA  log10 1 / T A  log10
  100 / TA  2 - log10 T 
• The last equation allows you to easily calculate
  absorbance from percentage transmittance data.

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• The Beer-Lambert Law
• the equation representing the law is
• Aebc
• Where
• A is absorbance (no units, since A log10 P0 / P
  )
• e is the molar absorbtivity with units of L
  mol-1 cm-1
• b is the path length of the sample - that is,
  the path length of the cuvette in which the
  sample is contained. We will express this
  measurement in centimetres.
• c is the concentration of the compound in
  solution, expressed in mol L-1

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• The ABS Photometer is an
• opto-electromechanical module which performs
  light-absorbance measurements on samples. Light
  from its standard source passes through the
  cuvette. Certain wavelengths of the light are
  absorbed in varying degrees depending on the
  composition of the sample in the cuvette. These
  wavelength absorptions are measured to provide an
  analysis of the cuvette contents.

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• Absorbance Photometry is a measurement technique
  for determining concentrations of substances in
  fluid samples. Substances, or substances reacted
  with reagents, absorb specific wavelengths of
  light. The amount of absorbance is a measure of
  the concentration in solution. In the 8-12
  wavelengths are available of which one or two are
  selected, the selection depending on the
  substance being measured. The 8-12 wavelengths
  are in the range from 340 to 660 nanometers (nm)
  as well as one (800nm) in the infrared range
  (visible light ranges from approximately 400 to
  700nm). A software algorithm calculates the
  concentration of the substance in solution
  depending on the measured absorbance
  characteristics.

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Block diagram
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Sample reading

• Method for determination of glucose in whole
  blood and cuvette and photometer for carrying out
  said method
• A sample of whole blood is contacted with a
  reagent which by chemical reaction with glucose
  in the sample brings about a detectable dye
  concentration change the size of which is
  determined as a measure of the glucose content of
  the sample. The sample is initially introduced
  undiluted in a microcuvette having at least one
  cavity for receiving the sample. The cavity is
  internally pretreated with the reagent in dry
  form, and the chemical reaction takes place in
  the cavity. Active components of the reagent
  comprise at least a hemolyzing agent for exposing
  glucose contained in the blood cells of the
  sample for allowing total glucose determination,
  and agents taking part in the chemical reaction
  and ensuring that the dye concentration change
  takes place at least in a wavelength range
  outside the absorption range of the blood
  hemoglobin. An absorption measurement is
  performed in said wavelength range directly on
  the sample in the cuvette.

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Chemistry block diagram
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Measurement Principles of CHEMISTRY ANALYZER

• Modern medicine relies heavily upon the analysis
  of body substances to evaluate and confirm a
  diagnosis. These analyses allow physicians - to
  prescribe a course of treatment for patients -
  to ascertain the stage of an illness and thus
  specify optimum treatments.
• Various body fluids may be analysed in different
  ways. Blood (both serum and plasma) and urine are
  the most commonly analysed, with blood being by
  far the more important of the two because of its
  various components.

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• Methods of analysis also differ widely, depending
  on purpose. The most useful and thus most
  commonly used are Ion Selective Electrode
  (ISE)analysis and Photometer analysis these are
  used in the Instrument.
• There are two measurement areas in the
  Instrument - ISE Module - Photometry Analyzer.

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Photometry Analyzer Photometer ABS Absorption
Analysis

• Photometry analysis methods are based upon
  measuring the amount of light absorbed by a
  substance. The measurements may be done directly
  on the substance or immediately after a chemical
  or enzymatic reaction. Chemical or enzymatic
  reaction methods require a reagent to be mixed
  with the sample before beginning the tests.

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• In certain situations, the concentration of a
  particular substance in a sample could be
  established by direct measurement of the amount
  of light absorbed. More often, however, the light
  absorption properties of a substance are
  inadequate for analysis purposes because
  interfering elements in the samples make them
  opaque. Thus samples normally are first mixed
  with a reagent (or sometimes two or three in
  sequence). The reagents are targeted to react
  with the substance to be measured only.

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• If the reagent is a chemical, the reaction
  converts the substance (or the reagent) into a
  specific quantity of dye product which can then
  be measured. This method may be used for defining
  most biologically significant metabolites in
  solution
• The concentration of a metabolite in solution may
  also be established using enzymatic techniques.
  In this method the enzyme converts a substance
  into a defined quantity of another product, which
  can be easily measured using photometric methods.
  This method is often the only one possible for
  complex biological mixtures.
• In a similar way enzymes in a sample can also be
  measured. Tracking enzyme levels in body fluids
  is an important diagnostic tool in modern
  medicine

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Photometric Measurements

• The normal sequence for a photometric measurement
  procedure consists of
• Transfer (pipette) a known quantity of sample
  into a cuvette
• Transfer appropriate reagent into the cuvette
• Ensure that sample and reagent are thoroughly
  mixed
• Add and thoroughly mix any additional (e.g.,
  starting) reagents required.
• Carry out photometric measurements
• Calculate and evaluate results

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• Concentration levels are calculated using the
  formula
• Concentration Absorption x Factor
• The multiplying factor is dimensioned depending
  on - Wither the absorption is at the absolute
  level of absorption - The change in
  absorption between the start and end of the test
  - The rate of change in absorption with respect
  to time.

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• Calculation of a factor may be based on
  information provided on the Reagent. Alternately,
  it may be determined by carrying out one or more
  calibration runs using a calibrator or a standard
  substance instead of a real sample. In such
  situations, the calibration run is repeated
  periodically (e.g. every five hours or at
  beginning of day BOD), or when a new Reagent
  is used.
• A calibrator is a serum-based substance that is
  used for calibrating several points for differing
  analysis tests.

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Kinetic or Enzymatic Measurements

• Kinetic or enzymatic Measurement procedures
  involve repeated photometric measurements at
  defined time intervals. For these measurements
  (also called continuous measurements),
  measurement values are obtained from a series of
  known standards and plotted using an x-y
  coordinate system.
• Each value represents a specific point in time (x
  axis) versus its corresponding absorbance value
  (y axis). Connecting the points producesa curve.

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• Values obtained from patient samples are compared
  against the "standard" curve to determine the
  concentration of enzyme in the sample. This curve
  may not necessarily be linear. The results may
  also be distributed. In some cases the actual
  change in value of absorption is known (i.e. the
  operator enters the measurement range). It is
  then only necessary to measure the time for the
  absorption to change from one value to the other.
  This provides the rate of change associated with
  the particular sample.
• Alternatively, it may be necessary to evaluate
  the slope of the curve where it is most linear.
  Here a linear regression process is applied to
  all data points to establish the required value
  of change in absorption with respect to change in
  time.

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End Point Procedures

• End point procedures look for a value at the end
  of a test. The rate of change in absorption with
  respect to time may be variable from test to test
  and therefore not suitable for measurement
  purposes. In this case it may be possible to
  establish the level of concentration of a
  substance by measuring the difference in
  absorption between the start and the end of a
  test (discontinuous measuringprocedure.(
• Here, time is not used in the calculation, but is
  normally fixed at a given value known to be
  sufficient to allow the full change in absorption
  due to reagent action. This type of procedure is
  typically used for slower reactions

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Application

• GGT
• Alkaline phosphatase
• Magnesium
• Osmolality
• Urate
• Iron
• Transferrin
• Total protein
• Globulins
• Glucose
• C-reactive protein
• (HBA1C) Glycohemoglobin

• Sodium (ISE)
• Potassium (ISE)
• Chloride (ISE)
• Bicarbonate
• Urea
• Creatinine
• Calcium
• Phosphate
• Albumin
• Bilirubin
• AST
• ALT

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• Creatinine. (Also known as Creatine
  phosphokinase, CK and CPK) is an enzyme which is
  very useful for diagnosing diseases of the heart
  and skeletal muscle. This enzyme is the first to
  be elevated after a heart attack. If CPK is high
  in the absence of heart muscle injury, this is a
  strong indication of skeletal muscle disease.
  Most creatinine is produced in the muscle, heart
  and brain. Creatinine is a water-soluble waste
  product largely from muscle breakdown that is
  excreted via the kidney tubules. Creatinine is
  not affected by the amount of urine produced and
  excreted. When creatinine breaks down it gives us
  energy because it acts as an enzyme important in
  the process of forming ATP (that very basic
  process that gives us energy. (

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• Uric Acid is a breakdown product of nucleic acids
  normally excreted in urine. Nucleic acids make up
  the components of DNA and RNA in our bodies.
• Uric Acid Too High Rule out gout, arthritis,
  kidney problems leukemia, lymphoma, polycythemia,
  acidosis, psoriasis, hypothyroidism, eclampsia,
  multiple meyeloma, pernicious anemia, tissue
  necrosis, inflammation, and the use of some
  diuretics.
• Uric Acid Too Low Rule out uricosuric drugs
  (drugs that break down uric acid and assist it to
  leave via the urine--as your pharmacist on this),
  too much allopurinol (the drug used in the
  treatment of gout), Wilson's Disease (a genetic
  disease of the liver which allows copper to build
  up to toxic levels), and large doses of Vitamin C.

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Unit Upper limit Lower limit Patient type Test
mmol/L or mEq/L 145-147 135-137   Sodium (Na)
mg/dl 33 - 34 31 - 32   Sodium (Na)
mmol/L or mEq/L 5.0-5.1 3.5-3.6   Potassium (K)
mg/dl 20 14   Potassium (K)
mmol/L or mEq/L 105-107 95-98   Chloride (Cl)
mg/dl 370 340   Chloride (Cl)
mOsm/kg 295-296 275-280   Osmolality
mOsm/l Slightly less than osmolality Slightly less than osmolality   Osmolarity
mmol/L 3.0-7.0 1.2-3.0   Urea
mg/dL 18-21 7   Urea
mmol/L 0.48 0.18   Uric acid
mg/dL 7 2 Female Uric acid
mg/dL 8.5 2.1 Male Uric acid
µmol/L 118 68 male Creatinine
mg/dL 1.3 0.8 male Creatinine
µmol/L 98 68 female Creatinine
mg/dL 1.1 0.8 female Creatinine
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Units

• Reagent Arm
• Reagent Rotor
• Measuring Unit
• Sample Rotor
• Syringes
• Sample Arm
• Cuvette Rotor
• Mixer
• PC Monitor Table
• Washing unit
• ISE
• Cooling Unit

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Cuvette Rotor
Sample Needle
PC Monitor Table
Reagent Needle
Reagent Rotor
Syringes
Sample Rotor
Measuring Unit
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Reagent and Sample Arm

• A Vertical stepper Motor
• B Motor Holder
• C Rod
• D Horizontal stepper Motor
• E optical Sensor
• F Encoder Disc

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Measuring Unit
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Sample Rotor

• A Sample Holder
• B - stepper Motor
• C - optical Sensor
• D - Encoder Disc
• E Timing Belt

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Syringes

• A Sample / Reagent Syringe
• B Valves
• C stepper Motor

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Reagent Rotor

• A Reagent Rotor
• B Stepper Motor
• C Encoder disc
• D Optical Encoder
• E Timing Belt

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Cuvette Rotor

• A Cuvette rotor
• B Peltier elements
• C Stepper motor

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Mixer
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Washing unit
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BASIC ISE
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• The Ion-Selective Electrode (ISE) module makes
• quantitative determinations of the following
  electrolytes
• Sodium
• Potassium
• Chloride
• Lithium
• The ISE module performs direct assays on
  undiluted serum or plasma
• samples (sodium, potassium, chloride, and
  lithium).
• It performs indirect assays on diluted serum or
  plasma or urine samples (sodium, potassium, and
  chloride only).

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• The concentration of ions in solution may be
  determined by using ion sensitive electrodes. The
  principle components of an ISE system are
  Sensing electrode (half cell( Reference
  Electrode (half cell( Readout device The
  measurement principle is based on the interaction
  between moveable free ions in a sample solution
  and an active sensing unit (ion selective sensing
  electrode)
• An ion-selective membrane separates the sample
  solution, where the electrolyte concentration is
  unknown, from the electrode electrolyte, where
  the concentration is known.

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• The membrane itself consists of a specific type
  of material which is able to react with one type
  of ions. Each ion selective electrode is
  equipped with a different type of membrane.
• Sodium - Glass membrane Potassium - Plastic
  membrane Chloride - Plastic membrane Lithium -
  Plastic membrane

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ISE Theory

• Chemical compounds in solution may behave in one
  of two ways. In one group, the structure of the
  molecules may remain intact or un-dissociated.
  These are non- electrolytes.
• The other group of compounds dissociate in
  solutions to form ions. This process is referred
  to as ionization and compounds that behave this
  manner are known as electrolytes.
• Ions carry an electrical charge. For example,
  sodium chloride (table salt) dissolved in water
  provides sodium ions (Na ) and chloride ions
  (Cl- ). The sodium ion carries a positive charge
  and the chloride ion carries a negative charge.

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• Measurement Modes
• There are three measurement modes
• Direct Mode The sample (plasma or serum),
  control, and standard
• solutions are used undiluted, the sample volume
• pipetted is 97 µL.
• Indirect Mode The sample (plasma or serum),
  control, and standard
• solutions are diluted with system water, the
  sample
• volume pipetted is 20 µL.
• Urine Mode The sample (urine), control, and
  standard solutions are
• diluted with system water, the sample volume
  pipetted
• is 20 µL.
• The dilutions and the mixing are performed
  automatically in the ISE tower.

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• Main Calibration
• Sodium, potassium, and chloride are calibrated
  with a two-point calibration
• using ISE Solutions 1 and 2
• One-Point Calibration
• The ISE module automatically performs a one-point
  calibration with the corresponding ISE
  calibrator, depending on the measurement mode.
• For example, in direct mode, the ISE Calibrator
  Direct is used for the one-point
• calibration.

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• Electrode Service
• Electrode Service is a service procedure that is
  performed automatically at the
• Begin-of-Day. While Electrode Service is taking
  place, the ISE module cannot
• carry out any tests.
• During electrode service
• The electrodes are cleaned with ISE
  Deproteinizer to prevent protein
• build up on the electrodes and in the tubing.
• The surface of the sodium electrode is etched
  (with the etcher solution
• located on the ISE rack).
• Electrodes are activated. The activator is
  pipetted from the ISE rack into
• the ISE tower and drawn through the measuring
  channel (performed
• twice), afterwards the electrodes are rinsed with
  ISE calibrator direct.

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ANALYTICAL MEHOTDS

• Calibration is the process that establishes the
  relation between one or more
• measured rates and the corresponding
  concentration of the calibrator. The
• measured rates are derived from instrument
  measurements, for example
• absorbance values.
• Calibrators are often human-based serum pools
  with known amounts of
• added analytes.
• Calibration interval
• Calibrations are performed at regular intervals
  (defined in Configuration /
• Tests /General) to compensate for changes over
  time in reagents and in the
• measurement systems.
• Calibrations also have to be performed when
  certain physical events occur.
• These include
• o A change in reagent.
• o A change in a defined interval.
• o Other physical changes, such as changing the
  photometer lamp.

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• Linear calibrations Tests for most enzymes and
  substrates have linear calibration curves, which
• require only a two-point calibration.
• K C2 C1/A1 - B
• C2 Concentration of standard solution 2
• C1 Concentration of standard solution 1
• A2 absorbance of standard solution 2 10?4
• B absorbance of standard solution 1 10?4

• CX K (X STD (1) C1) IFa IFb
• Cx Concentration of sample
• C1 Concentration of standard solution 1
• C2 Concentration of standard solution 2
• STD1 Absorbance or Absorbance change rate of
  standard solution 1
• STD2 Absorbance or Absorbance change rate of
  standard solution 2
• K Calculation factor
• IFa,iFb Instrument constant

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Non-linear calibrations

• Non-linear calibrations Tests for most specific
  proteins, drugs of abuse testing (DAT) and
  therapeutic
• drug monitoring (TDM) have non-linear
  calibrations.
• For a non-linear calibration, between four and
  six calibration points are used,
• depending on the calibration mode.

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SAFETY REGULATIONS

• Biological Safety
• Samples analyzed with this instrument may contain
  potentially biohazardous material such as
  bacterial, fungal or viral agents.
• Follow these General Rules at all times
• Wear protective gloves and clothing
• Never eat, drink or smoke while working in a
  laboratory environment
• Wash your hands after working
• During service work, keep your hands and fingers
  away from your mouth, nose and eyes

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• Electrical and Mechanical Safety
• To avoid the possibility of short circuits and
  resultant burns or even electrocution, remove all
  jewelry from your hands, arms and neck when
  servicing this equipment.
• Never remove or disconnect the protective ground
  (earth) conductor inside or outside the
  instrument. Doing this can lead to lethal shocks.
  
• Never place head, hands, tools or any other
  object in the transfer area when the instrument
  is operational. Failure to follow this
  instruction can result in severe injury and
  instrument damage.

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Trouble shooting
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Thank you for your attention