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					 Medical Laboratory
    Third Year
     Dr Fadhl Alakwa
  Biomedical Department
Blood (Purpose and components)
• Blood is the fluid that circulates
  trough the heart, arteries, veins
  and capillaries carrying
  nourishment, electrolytes,
  hormones, vitamins, antibodies,
  heat and oxygen to body tissues
  and taken a way waste matter
  and carbon dioxide.
• Blood is composed of cells and
Blood cell Portion
• Red blood cells
• White blood cells
• Platelets
Red blood cells
•   Disc-shaped cells
•   Contain no nucleus
•   Live 120 days
•   Number 4.5 to 5.5 million cells/mm3
•   Each RBC contains 4 iron atoms in a
    structure known as the hemoglobin
White blood cells
•   Amoeba like cells
•   Contain a nucleus
•   Live 20 days
•   Number 6 to 10 thousands cells/mm3
•   They are present in the lymph fluid and
    engulf invading bacteria and foreign
    substances to destroy the invaders’ effect.
•   They are cell fragments
•   Contain no nucleus
•   Number 200 to 800 thousands cells/mm3
•   Blood coagulation and clotting
Blood plasma
•   Plasma proteins
•   Plasma nutrients-energy-storing
•   Regulatory and protective substances
•   Plasma electrolytes
•   Metabolic waste substances
Plasma proteins
• Albumins
• Fibrinogen and prothrombin
• Globulin
Plasma nutrients-energy-storing
• Glucose (blood sugar)
• Lipids (fats)
• Amino acids (Proteins for tissue growth)
Regulatory and protective substances

• Enzymes
• Hormones
• Antibodies
Plasma electrolytes-acid-base
• Na+
• K+
• Cl-
  Metabolic waste substances
  • Urea
  • Uric
Purpose of M. L. I.
 The purpose of medical laboratory
 instrumentation is to provide a means of
 measuring required substances and
 metabolic waste products in urine and
  Instrumental Analysis is the Base for All the
  Modern Sciences

Instrumental Analysis will give quick answers on (1) what species is a

certain system (qualitative) and (2) How many of them (quantitative).

  Analytical chemistry is critical to our understanding of
  biochemistry, medicinal chemistry, geochemistry, environmental
  science, atmospheric chemistry, materials science, metallurgy,
  biology, pharmacology, agricultural science, food science,
  geology, and other fields.
Qualitative analysis
• Qualitative analysis is the branch of
  analytical chemistry that is concerned with
• such as “What makes this water smell
  bad?”, “Is there gold in this rock sample?”,
  “Is this sparkling stone a diamond or cubic
  zirconia?”, “Is this plastic item made of
  polyvinyl chloride, polyethylene or
  polycarbonate?”, or “What is this white
Quantitative Analysis
• When qualitative analysis is completed, the
  next question is often “How much of each
  or any component is present?” or “Exactly
  how much gold is this rock?” or “How
  much of the organochlorine pesticide
  dieldrin is in this drinking water?”
• The determination of how much is
  quantitative analysis.
undergraduate instrumental analysis page 9,10,11,12
            Basics of Instrumental Analysis

   Stimulus                                            Response

 Energy Source                 Sample                 Analytical Information

Input transducer   Data domain of       Information
                   Transduced           processor             Readout
Basics of Instrumental Analysis
• All instruments measure some chemical or
  physical characteristic of the sample, such as how
  much light is absorbed by the sample at a given
  wavelength, the mass-to charge ratio of an ion
  produced from the sample, or the change in
  conductivity of a wire as the sample passes over it.
  A detector of some type makes the measurement
  and the detector response is converted to an
  electrical signal. The electrical signal should be
  directly related to the chemical or physical
  property being measured and that should be
  related to the amount of analyte present.
Selecting Analytical Instruments
   In order to select an analytical method intelligently, it is essential
to define clearly the nature of the analytical problem. Such a
definition requires answers to the following questions:

1. What accuracy is required?
2. How much sample is available?
3. What is the concentration range of the analyte?
4. What components of the sample will cause interference?
5. What are the physical and chemical properties of the
  sample matrix?
6. How many samples are to be analyzed?
               Precision and Accuracy

Not precise       Not precise    Precise        Precise
Not accurate      But accurate   And accurate   But not accurate
Statistics: be care the next term
Signal &Noise
• Merely providing data to other scientists is
  not enough; the analytical chemist must be
  able to interpret the data, and communicate
  the meaning of the results, together with the
  accuracy and precision (the reliability) of
  the data, to scientists who will use the data.
S/N ratio
Every analytical measurement is made up of two components:

                   signal and noise

Signal: carries information about the analyte

Noise: made up of extraneous information that is unwanted
because it degrades the accuracy and precision of an analysis
and also places a lower limit on the amount of analyte that can
be detected.
Signal-to-noise ratio
     Actual recording                 Average

Figure 5-1 Effect of noise on a current measurement: (a)
experimental strip-chart recording of a 0.910-15 A direct
current, (b) mean of the fluctuations. (Adapted from T.
Coor, J. Chem. Educ., 1968,45,A594. With permission.)
        Calculation of signal/noise ratio
        The following data were obtained for a voltage
measurement, in V, on a noise system: 1.37, 1.94, 1.35,
1.47, 1.41, 1.63, 1.54, 0.78. Assuming the noise is random,
what is the signal to noise ratio?
     x = 1.436           N = 0.232              S/N= 6.19

If the maximum and the minimum are used for the calculation:
N= (1.94-0.78)/5 = 0.232

As a general rule, it becomes impossible
to detect a signal when the signal-to-
noise ratio becomes less than 3.

    Detection limit considerations
Signal/noise calculation

Noise types

Noise reduction

Multiple measurements and S/N
Noise Types

       Noise from a given source may or may not be random.
The sign and magnitude of the instantaneous voltage deviation
from the mean value are unpredictable for random noise.

        random noise

      nonrandom noise,
   Noise can also be classified as fundamental or

   Fundamental noise arises from the particle nature of light
and matter and can never be totally eliminated.

     Nonfundamental noise, often called excess noise, is due
to imperfect components and instrumentation; theoretically,
it can be eliminated completely.

    Random noise can be fundamental or nonfundamental
noise, but nonrandom noise is never fundamental noise.

     The noise observed in a signal at any instant is due to
the summation of the fluctuations caused by a large number
of random and nonrandom noise sources
                  White Noise
For white or Gaussian noise, the magnitude of the noise
power P(e) is independent of frequency. White noise is
random noise and is usually fundamental noise, although
it can arise because of nonfundamental causes.

        Figure. Noise power spectrum (NPS)
Sources of noise in instrumental analysis:
1. Chemical noise:
    undetected variations in temperature, pressure,
humidity, and other factors.

2. Instrumental noise:
complex composite cannot be fully characterized

five different kinds of noises:
     Thermal noise, Johnson noise
     Shot noise, 1/f noise
     flicker noise
     environmental noise
      white noise
Environmental noise

    Figure 5-3 Some sources of environmental noise in a
    university laboratory. Note the frequency dependence
    and regions where various types of interference occur.
Environmental noise

environmental noise is a frequency-dependent,
Nonfundamental noise.

       the amplitude, frequency, and phase are predictable.

       The most common interference noise in the United
States is 60-Hz noise from ac power lines;

        Some interference noise, often called impulse noise,
is correlated noise, that is random in time. Examples
are noise spikes generated by turning instruments on
and off, spikes on the ac power line, and radio-frequency noise
from spark gaps in lasers.
signal/noise calculation

Noise types

noise reduction

multiple measurements and S/N
     Signal/noise Ratio Enhancement
       Two general methods are available for improving
the signal-to-noise ratio of an instrumental method,
namely hardware and software.
Some Hardware Devices for Noise Reduction

                          ANALOG FILTERING

                          Figure 5-5 Use of a low-pass
                          filter with a large time
                          constant to remove noise
                          from a slowly changing dc

        Direct amplification of a low-frequency or dc signal
particularly troublesome because of amplifier drift and
flicker noise. Often, this l/f noise is several times larger than
the types of noise that predominate at higher frequencies.

        For this reason, low-frequency or dc signals from
transducers are often converted to a higher frequency, where
1/f noise is less troublesome.

       After amplification, the modulated signal can be freed
from 1/f noise by filtering with a high pass filter; demodulation
and low pass filtering will result in a suitable signal for output.
 S/N Advantages

FIGURE 5-8 Advantage of modulation. Modulation is used to encode the
signal information at the modulation frequency f m. The background noise
observed is less at frequency f m (region B) than at dc frequencies (region
A) for the same noise equivalent bandpass (f). The rms noise is
proportional to the square root of the area under the NPS defined by f. To
discriminate against 1/f noise, the signal processor is adjusted to respond
only to the signal encoded at f m and the noise in the bandwidth centered
around f m . It is important to choose f m to be high enough that the 1/f noise
has fallen off and to be at a frequency where interference noise is negligible
(f m should not be in region C).
Software methods

       ensemble averaging
Figure 5-10 Effect of signal averaging. Note that the
vertical scale is smaller as the number of scans
increases. The signal-to-noise ratio is proportional to .
Random fluctuations in the noise tend to cancel as the
number of scans increases. but the signal accumulates;
thus, S/N increases.
The signal-to-noise ratio S/N for the signal average is given by

Thus, the signal-to-noise ratio increases by the square root of
the number of times the data points are collected and

To realize the advantage of ensemble averaging and still
extract all of the information available in an analyte wave
form, it is necessary to measure points at a frequency that is
at least twice as great as the highest frequency component of
the wave form. Much greater sampling frequencies, however,
provide no additional information but include more noise.
Signal/noise calculation

Noise types

Noise reduction

Multiple measurements and S/N
Improve the signal/noise ratio by more
        The following data were obtained for a voltage
measurement, in V, on a noise system: 1.37, 1.94, 1.35,
1.47, 1.41, 1.63, 1.54, 0.78. Assuming the noise is random,
what is the signal to noise ratio? How many measurements
would have to be averaged
to achieve a S/N of 20?
Concentration Unit
• Many analytical results are expressed as the concentration of the
  measured substance in a certain amount of sample. The measured
  substance is called the analyte.
• Commonly used concentration units include molarity (moles of
  substance per liter of solution), weight percent (grams of substance per
  gram of sample 100%), and units for trace levels of substances.
• One part per million (ppm) by weight is one microgram of analyte in a
  gram of sample, that is, 1 x 10-6 g analyte/g sample. µg/g
• One part per billion (ppb) by weight is one nanogram of element in a
  gram of sample or 1 x 10-9 g analyte/g sample.
• parts per trillion of the element, that is, picograms of element per gram
  of sample (1 x10-12 g analyte/g sample).
Concentration Unit?
• To give you a feeling for these quantities, a
  million seconds is 12 days (11.57 days, to
  be exact). One part per million in units of
  seconds would be one second in 12 days.
• A part per billion in units of seconds would
  be 1 s in 32 years, and one part per trillion
  is one second in 32,000 years.
• A sample may be homogeneous, that is, it
  has the same chemical composition
  everywhere within the sample. Like the salt
• Many samples are heterogeneous; the
  composition varies from region to region
  within the sample.
Medical laboratory department
• Facilities
• Personnel
• Equipment
• Must includes a clean, safe surrounding
  with a special area for sterilization of
  contaminated blood urine samples and
• Sufficient storage and cleaning areas must
  be designated
• Physician
• Medical technologist (equipment operator)
• Supervisor
• Glassware, centrifuges, suction devices
• Colorimeter
Is an optical devise that measures the color
  concentration of a substance in solution
• Flame photometer
Is an optical electronic devise that measures
  the color intensity of substance that have
  been aspirated into a flame (sodium and
• Spectrophotometer
Is optical device that measure light absorption
  at various wavelengths for a given liquid
• Blood cell analyzer
Is a device to measures the number of red and
  white blood cells per scaled volume.
The aperture impedance and flow cytometery
• Ph/ blood gas analyzer
Is a device which measure blood Ph, Po2,
• Chromatograph and Autoanalyzer
Is a electromechanical device used to
  separate, identify, and measure the
  concentration of substances in a liquid
• Computer based record and operation
 Introduction to Spectroscopy
• Spectroscopy is the
  science which study
  the interaction of
  radiation with matter.
• the study of molecular
  structure and
  dynamics through the
  absorption, emission,
  and scattering of light.
What is Electromagnetic Radiation?
• Visible light that comes from your lamb and
  radio waves from your radio station.
• Example: Radio waves, Microwaves, IR,
  Visible, UV, X-ray, Gamma ray.
What is Electromagnetic Radiation?

        E = hn         n=c/l
X-Ray           UV           Visible           IR       Microwave

        200nm        400nm             800nm        100,000nm

The Nature
of Light
radiation is
 viewed as both a wave
and a particle
wave-particle duality
     Understanding the nature of light

1. Light is composed of particles
2. Light is wave
  a. General concepts of Wave
      (wavelength, frequency, velocity, amplitude)
  b. Properties of Wave
       I. Diffraction & Coherent Radiation
      II. Transmission & Dispersion
      III. Refraction: Snell’s Law
3. Black Body Radiation and photoelectric effect
              wave-particle duality
4. Interaction between electromagnetic radiation
   and matter for spectroscopy: scattering, absorption,
   and emission
Light travels in a straight line
         Light is consists of small particles

The Thomas Young’s Experiment (1801)

    Interference phenomenon: Light is wave!
Light is Electromagnetic Wave

              What is a wave?!


           c            velocity
                   Velocity (300,000,000 meters/sec)
Frequency        =     Propagation

               l Wavelength (meters)
                        Wave Parameters
        The amplitude A of the sinusoidal wave is shown as the
length of the electric vector at a maximum in the wave.

        The time in seconds required for the passage of successive
maxima or minima through a fixed point in space is called the
period, p, of the radiation.

        The frequency, n, is the number of oscillations of the field
that occur per second and is equal to l/p.

        Another parameter of interest is the wavelength, l, which is
the linear distance between any two equivalent points on successive
waves (e.g., successive maxima or minima).
angstrom:       10 -10 m nanometer: 10 -9 m
micrometer: 10 -6 m millimeter: 10 -3 m
Equation of wave motion
• Y =a sin(wt-kx+Θ)
• Displacement due to wave at any distance x
  and time t
• a maximum displacement
• W=2pi*f (angular velocity)
• k =2 pi /wavelength (propagation constant)
• Θ phase angle
Equation of wave motion
• Mechanical wave
• Sound wave
• Electromagnetic wave
Electromagnetic Energy
• Light is composed of particles ”Photons”
• E =hf =hc/λ h= 6.626x10^-34 j.s (Plank
• Photon energy unit is (e.v)
• Energy gained by one electron when
  accelearted by potential difference of one
• e.v=1.6x10^-19 coulomb x 1 volt=
  =1.6x10^-19 Joule
• Component >> atom like Iron {FE}
• Compound >> Molecular like Sugar
  {CHO} {more than one atom}
Atom                           Neutron

  Proton                             Electron

Mass number
      A=# protons + Neutrons      Uranium
      Z = # electrons               U
       Atomic Number              92
       Periodic table
Isotopes the same Z and different A

            1       Hydrogen     One proton
    1           2                 One proton
                     Deuterium    One neutron
                3                 One proton
            H       Tritium       Two neutrons

             When Light Strikes Matter…

and refraction
Excitation methods:
•   (i) EM radiation
•   (ii) Spark/discharge/arc
•   (iii) Particle bombardment (electrons, ions... )
•   (iv) Chemiluminescence (exothermic
    chemical reaction generates excited
Absorption Spectra

Spectra Plot of
Absorbance vs.
Emission Spectra

Spectra Plot of
intensity vs.
Question page 33 Example 2
• Given that the ionization potential of
  hydrogen atom is 13.6 volt and the energy
  level of any………..

• Home work 1 ,2, 3 pages 39 and 40

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