Medical Laboratory
Instrumentation
2010-2011
Third Year
Dr Fadhl Alakwa
www.Fadhl-alakwa.weebly.com
UST-Yemen
Biomedical Department
http://fadhl-alakwa.weebly.com/
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
plasma.
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.
Platelets
•
•
•
•
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
http://en.wikipedia.org/wiki/Reference_ranges_for_common_blood_tests
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
blood.
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
questions
• 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
powder?”
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
Energy Source
Input transducer
Response
Analytical Information
Sample
Data domain of
Transduced
information
Information
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 accurate
Not precise
But accurate
Precise
And accurate
Precise
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
N
As a general rule, it becomes impossible
to detect a signal when the signal-tonoise 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
non-fundamental.
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
GROUNDING AND SHIELDING
DIFFERENCE AND INSTRUMENT AMPLIFIERS
ANALOG FILTERING
MODULATION
ANALOG FILTERING
Figure 5-5 Use of a low-pass
filter with a large time
constant to remove noise
from a slowly changing dc
voltage.
MODULATION
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 .
n
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
averaged.
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
measurements
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.
Terminology
• A sample may be homogeneous, that is, it
has the same chemical composition
everywhere within the sample. Like the salt
water.
• Many samples are heterogeneous; the
composition varies from region to region
within the sample.
Medical laboratory department
• Facilities
• Personnel
• Equipment
Facilities
• Must includes a clean, safe surrounding
with a special area for sterilization of
contaminated blood urine samples and
equipment
• Sufficient storage and cleaning areas must
be designated
Personnel
• Physician
• Medical technologist (equipment operator)
• Supervisor
Equipment
• 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
potassium)
Equipment
• Spectrophotometer
Is optical device that measure light absorption
at various wavelengths for a given liquid
sample.
• 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
Equipment
• Ph/ blood gas analyzer
Is a device which measure blood Ph, Po2,
Pco2
• Chromatograph and Autoanalyzer
Is a electromechanical device used to
separate, identify, and measure the
concentration of substances in a liquid
medium.
• Computer based record and operation
system
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
200nm
Visible
400nm
IR
800nm
WAVELENGTH(nm)
Microwave
100,000nm
The Nature
of Light
Electromagnetic
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
Newton
The Thomas Young’s Experiment (1801)
Interference phenomenon: Light is wave!
Light is Electromagnetic Wave
Maxwell
(1864)
What is a wave?!
amplitude
Harmonic
wave:
wavelength
frequency
or
wavelength
velocity
=
wavelength
l Wavelength
(meters)
c
frequency
n=
Frequency
(Hertz)
Velocity (300,000,000 meters/sec)
Propagation
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
constant)
• Photon energy unit is (e.v)
• Energy gained by one electron when
accelearted by potential difference of one
volt
• e.v=1.6x10^-19 coulomb x 1 volt=
=1.6x10^-19 Joule
Matter
• Component >> atom like Iron {FE}
• Compound >> Molecular like Sugar
{CHO} {more than one atom}
•
Atom
Proton
Mass number
A=# protons + Neutrons
X
Z = # electrons
Atomic Number
Periodic table
Neutron
Electron
Uranium
238
U
92
Isotopes the same Z and different A
1
Hydrogen
H
1
2
H
1
3
H
1
Deuterium
Tritium
One proton
One proton
One neutron
One proton
Two neutrons
When Light Strikes Matter…
Transmission
Reflection
and refraction
Interference
Scattering
Excitation methods:
•
•
•
•
(i) EM radiation
(ii) Spark/discharge/arc
(iii) Particle bombardment (electrons, ions... )
(iv) Chemiluminescence (exothermic
chemical reaction generates excited
products
Absorption Spectra
Absorption
Spectra Plot of
Absorbance vs.
wavelength
called
absorption
spectrum.
Emission Spectra
Emission
Spectra Plot of
emission
intensity vs.
wavelength
called
emission
spectrum.
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