MEDICAL DIAGNOSIS USING CHEMICAL TESTS

•
Most of the pathological processes result in chemical changes in the
internal environment of the human body. These changes can
generally be detected by the analysis of various samples taken from
the body.
•
The analysis not only helps in the diagnosis of various ailments but
also in determining the progress of treatment and for making a
prognosis.
•
Samples taken from the body are analyzed in three different areas
within the clinical laboratory set up.
•
These are:
• Chemistry section deals with the analysis of blood, urine, cerebrospinal fluid
(CSF) and other fluids to determine the quantity of various important
substances they contain.
•
The most common substance for analysis from the body is blood. This is
because the blood carries out the most important function of
transportation and many pathological processes manifest themselves as
demonstrable changes in the blood.
•
Deviations from the normal composition of urine also reflect many
pathological processes. The liquid part of the blood—the blood plasma,
and the formed elements—the blood cells are analyzed during a chemical
examination.
•
The blood plasma accounts for about 60% of the blood volume and the
blood cells occupy the other 40%. The plasma is obtained by centrifuging a
blood sample.
•
During centrifugation, the heavy blood cells get packed at the bottom of
the centrifuge tube and the plasma can thus be separated. The plasma is a
viscous, light yellow liquid, i.e. almost clear in the fasting stage.
Spectrophotometry
•
Spectrophotometry is the most important of all the instrumental
methods of analysis in clinical chemistry.
•
This method is based on the absorption of electromagnetic radiation
in the visible, ultraviolet and infrared ranges.
•
According to the quantum theory, the energy states of an atom or
molecule are defined and change from one state to another, would,
therefore, require a definite amount of energy.
•
If this energy is supplied from an external source of radiation, the
exact quantity of energy required to bring about a change from one
given state to another will be provided by photons of one particular
frequency, which may thus be selectively absorbed.
•
The study of the frequencies of the photons which are absorbed
would thus provide information about the nature of the material.
•
Also, the number of photons absorbed may provide information
about the number of atoms or molecules of the material present in a
particular state. It thus provides us with a method to have a
qualitative and quantitative analysis of a substance.
•
Molecules possess three types of internal energy—electronic,
vibrational and rotational.
•
When a molecule absorbs radiant energy, it can increase its internal
energy in a variety of ways.
•
The various molecular energy states are quantized and the amount of
energy necessary to cause any change in any one of the above energy
states would generally correspond to specific regions of the
electromagnetic spectrum.
•
Electronic transitions correspond to the ultraviolet and visible regions,
vibrational transitions to the near infrared and infrared regions and
rotational transitions to the infrared and far-infrared regions.
•
The method based on the absorption of radiation of a substance is
known as Absorption Spectroscopy.
•
The main advantages of spectrometric methods are speed, sensitivity
to very small amounts of change and a relatively simple operational
methodology.
•
The time required for the actual measurement is very short and most
of the analysis time, in fact, goes into preparation of the samples.
•
The region in the electromagnetic spectrum which is normally used in
spectroscopic work is very limited.
•
Visible light represents only a very small portion of the electromagnetic
spectrum and generally covers a range from 380 to 780 mm.
Interaction of radiation with matter
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When a beam of radiant energy strikes the surface of a substance, the
radiation interacts with the atoms and molecules of the substance.
•
The radiation may be transmitted, absorbed, scattered or reflected, or it
can excite fluorescence depending upon the properties of the
substance.
•
The interaction however does not involve a permanent transfer of
energy.
•
The velocity at which radiation is propagated through a medium is less
than its velocity in vacuum.
•
It depends upon the kind and concentration of atoms, ions or
molecules present in the medium.
•
Various possibilities which might result when a beam of radiation
Interaction of radiation with matter
Typical spectrophotometer
The essential components are:
• A source of radiant energy, which may he a tungsten lamp, a xenon-
mercury arc, hydrogen or deuterium discharge lamp, etc.
• Filtering arrangement for the selection of a narrow band of radiant
energy. It could be a single wavelength absorption filter, an interference
filter, a prism or a diffraction grating.
• An optical system for producing a parallel beam of filtered light for
passage through an absorption cell (cuvette). The system may include
lenses, mirrors, slits, diaphragm, etc.
• A detecting system for the measurement of unabsorbed radiant
energy, which could be the human eye, a barrier-layer cell, phototube
or photo-multiplier tube.
Various components of a spectrophotometer type instrument
RADIATION SOURCE
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The function of the radiation source is to provide a sufficient intensity
of light which is suitable for making a measurement. The most
common and convenient source of light is the tungsten lamp.
•
This lamp consists of a tungsten filament enclosed in a glass envelope.
It is cheap, intense and reliable.
•
A major portion of the energy emitted by a tungsten lamp is in the
visible region and only about 15 to 20% is in the infrared region.
•
When using a tungsten lamp, it is desirable to use a heat absorbing
filter between the lamp and the sample holder to absorb most of the
infrared radiation without seriously diminishing energy at the desired
wavelength.
•
For work in the ultraviolet region, a hydrogen or deuterium discharge
lamp is used.
Optical filters
•
A filter may be considered as any transparent medium which by its
structure, composition or colour enables the isolation of radiation of a
particular wavelength.
•
For this purpose, ideal filters should be monochromatic, i.e. they must
isolate radiation of only one wavelength.
A filter must meet the following two requirements:
(a) high transmittance at the desired wavelength and
(b) low transmittance at other wavelengths.
•
However, in practice, the filters transmit a broad region of the
spectrum.
•
Filters can be broadly classified as absorption filters and interference
filters.
Monochromators
•
Monochromators are optical systems, which provide better isolation of
spectral energy than the optical filters, and are therefore preferred
where it is required to isolate narrow bands of radiant energy.
•
Monochromators usually incorporate a small glass of quartz prism or a
diffraction grating system as the dispersing media.
•
The radiation from a light source is passed either directly or by means of
a lens or mirror into the narrow slit of the monochromator and allowed
to fall on the dispersing medium, where it gets isolated.
•
The efficiency of such monochromators is much better than that of
filters and spectral half-bandwidths of I nm or less are obtainable in the
ultraviolet and visible regions of the spectrum.
Optical components
•
Several different types of optical components are used in the construction
of analytical instruments based on the radiation absorption principle.
They could be windows, mirrors and simple condensers.
•
The material used in the construction of these components is a critical
factor and depends largely on the range of wavelength of interest.
•
Normally, the absorbance of any material should be less than 0.2 at the
wavelength of use. Some of the materials used are:
• Ordinary silicate glasses which are satisfactory from 350 to 3000 nm.
• From 300 to 350 nm, special corex glass can be used.
• Below 300 nm, quartz or fused silica is utilized. The limit for quartz is 210
nm.
• From 180 to 210 nm, fused silica can be used, provided the
Photosensitive detector
•
After isolation of radiation of a particular wavelength in a filter or a
monochromator, it is essential to have a quantitative measure of its
intensity.
•
This is done by causing the radiation to fall on a photosensitive element,
in which the light energy is converted into electrical energy.
•
The electric current produced by this element can be measured with a
sensitive galvanometer directly or after suitable amplification.
•
Any type of photosensitive detector may be used for the detection and
measurement of radiant energy, provided it has a linear response in the
spectral band of interest and has a sensitivity good enough for the
particular application.
•
There are two types of photo-electric cells; photo-voltaic cells and photo-
emissive cells.
Sample holders
Liquids may be contained in a cell or cuvette made of transparent
material such as silica, glass or perspex. The faces of these cells through
which the radiation passes are highly polished to keep reflection and
scatter losses to a minimum.
Solid samples are generally unsuitable for direct spectrophotometry. It
is usual to dissolve the solid in a transparent liquid.
Gases may be contained in cells which are sealed or stoppered to make
them air-tight.
The sample holder is generally inserted somewhere in the interval
between the light source and the detector.
For the majority of analyses, a 10 mm path-length rectangular cell is
usually satisfactory.
Microprocessor based spectrophotometer
•
Since the advent of the microprocessor, their application has not only
been limited to processing of data from analytical instruments, but
has also been extended to control of instrument functions and digital
signal processing, which had been performed conventionally by
analog circuits.
•
This has resulted in improved performance, operability and reliability
over purely analog instruments.
•
A microprocessor, in a spectrophotometer, could be used for the
following functions:
• Control functions: Wavelength scanning, automatic light source
selection, control of slitwidth, detector sensitivity, etc.
• Signal processing functions: Baseline correction, signal smoothing,
calculation of % T, absorbance and concentration, derivative, etc.
Block diagram of a microprocessor controlled spectrophotometer
•
The diagram shows only the post-detector electronic handling and
drive systems, all controlled via a single microprocessor.
•
Once the operator defines such parameters as wavelength, output
mode and relevant computing factors, the system automatically
ensures the correct and optimum combination of all the system
variables.
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Selection of source and detector are automatically determined; any
filters introduced at appropriate points and sample and reference
cells are correctly managed in the sample area.
•
Output in the desired form (transmittance, absorbance,
concentration, etc.) is presented along with the sample identification.
Secondary routines such as wavelength calibration and self-tests
become available on demand.
•
For wavelength scanning, a stepper motor is used, which ensures
accurate and fast scanning.
•
The signal from the photo-detector is amplified in a preamplifier and
converted into digital form in an A-D converter.
•
The signals are differentiated into sample signal S, reference signal R
and zero signal Z and stored in the memory. From these values, the
microprocessor calculates the transmittance T = (S-Z/R-Z) and
absorbance = –Log T.
•
In order to obtain R or S values within a specified range, the
microprocessor provides control signals for slit-width and high voltage
for the photomultiplier.
•
Baseline compensation due to solvent and optical unmatching of cells,
which has been difficult with conventional instruments is conveniently
possible in microprocessor based systems.
•
Improvements have also been achieved in such functions as auto-
zero, expanding and contracting of the photometric scale, automatic
setting of wavelength as well as in ensuring repeatable and more
•
PC-based spectrophotometers are now commonly available.
•
In these instruments, wavelength drive, slit drive, filter wheel drive,
source selector, mirror drive, detector change and grating drive are all
carried out by stepper motors.
•
Stepping control is effected by the computer, with the pulse
frequency depending upon the individual scan speed.
•
The computer processes the data supplied by the microprocessor and
transmits it with the suitable format to the video display screen and
printer/plotter.
Selective Ion electrodes based electrolytes analyzer
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Over the past decade, the pH meter has been at the centre of a most
important change in the field of analytical measurements due to the
introduction of selective-ion electrodes.
•
As their name implies, these electrodes are sensitive to the activity of
a particular ion in solution and quite insensitive to the other ions
present.
•
As the electrode is sensitive to only one ion, a different electrode is
needed for each ion to be studied. Approximately 20 types of
selective-ion electrodes are presently available.
Ion-selective electrodes are classified into four major groups:
•
Glass Electrodes: The first glass ion-selective electrode developed is the
one sensitive to hydrogen ions. Glasses containing less than 1% of Al2O3
are sensitive to hydrogen ions (H+) but almost insensitive to the other
ions present.
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Glasses, of which the composition is Na2O–11%, Al2O3–18%, Si O2–71%
is highly selective towards sodium, even in the presence of other alkali
metals. Glass electrodes have been made that are selectively sensitive to
sodium, potassium, ammonium and silver.
•
Solid State Electrodes: These electrodes use single crystals of inorganic
material doped with a rare earth material. Such electrodes are
particularly useful for fluoride, chloride, bromide and iodide ion analysis.
•
Liquid-Liquid Membrane Electrodes: These electrodes are essentially
liquid ion-exchangers, separated from the liquid sample by means of a
permeable membrane.
•
Gas Sensing Electrodes: These electrodes respond to the partial pressure of
the gases in the sample. The most recent of these to be developed are the
gas sensing electrodes for ammonia and sulphur dioxide.
•
Ammonia or sulphur dioxide is transferred across a gas permeable
membrane, until the partial pressure in the thin film of filling solution
between the glass electrode membrane and the probe membrane equals
that in the sample.
•
The resultant pH change is measured by a combination pH electrode. A
potential is developed related to the partial pressure and hence the
ammonia or sulphur dioxide concentration is measured.
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Applications using ion-selective electrodes are many, most being time saving
and simple to use. The electrodes are now used for the continuous
monitoring of important electrolytes in the blood such as sodium,
potassium, calcium, chloride, etc.
The pH meter
•
A pH meter is a precise instrument that weighs the hydrogen-ion
movement in water-based suspensions, showing its acidity or
alkalinity expressed as pH.
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It is also called a “potentiometric pH meter” because it measures the
variation in electrical potential between a pH electrode and a
reference electrode.
•
The variation in electrical potential links to the acidity or pH of the
suspension.
•
This meter is used for experimentation, quality control, etc.
•
The word pH is acquired from “p,” the scientific figure for negative
logarithm, and “H,” the chemical symbol for Hydrogen.
pH Measurement
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The pH rate of a material is directly linked to the degree of the
hydrogen ion [H+] and the hydroxyl ion [OH-] concentrations.
•
The quantitative data rendered via the pH meter shows the ratio of
the movement of an acid or base in terms of hydrogen ion activity.
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If the H+ density is higher than OH-, the substance is acidic; i.e., the
pH amount is less than 7.
•
If the OH- intensity is higher than H+, the substance is basic, including
a pH value higher than 7.
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If identical quantities of H+ and OH- ions are present, the substance is
neutral, with a pH of 7.
pH Meter Working Principle
•
A pH meter is made of a few vital components such as Measuring
Electrode, Reference Electrode, Temperature Sensor.
•
The pH Meter estimates the voltage of an electrochemical cell and
based upon the Temperature Sensor defines the pH of a suspension.
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Most of the pH meters contain Combination Electrodes, in which the
electrodes and the Temperature Sensor are fabricated within a single
frame.
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The algebraic total of the potentials of the Measuring Electrode,
Reference Electrode, and the Liquid Junction is known as the overall
potential or the voltage.
•
The Reference Electrode contains a neutral solution such as Potassium
Chloride solution with a fixed concentration. It gives a stable voltage.
•
On the opposite, the potential of the Measuring Electrode depends
totally upon the pH of the suspension.
•
The potential variation (voltage) between a glass membrane of
Measuring Electrode and a Reference Electrode which is immersed in
the Sample Liquid to be examined is estimated.
•
When the two Electrodes are immersed into the Sample Suspension,
the ion-exchange process transpires wherein some of the Hydrogen
ions flow towards the outside surface of the Measuring Electrode and
displace some of the metal ions within it.
•
Likewise, some of the metal ions migrate from the Glass Electrode
toward the Sample Suspension. The responsiveness of the Reference
Electrode potential to variation in pH is negligible or it is unaffected by
variations in pH and therefore produces a stable voltage.
•
on-exchange processes additionally takes place on the interior surface
of the Glass Electrode from the sample suspension.
•
This generates a potential variation (Hydrogen- ion activity) among
them. The Liquid Junction potential is normally minute and almost
constant which es sentially depends on the intensity of the ions in the
sample suspension.Every three potentials are summed up and ranked
by High Impedance Voltmeter.
•
The potential voltage generated beyond the Glass Electrode
membrane is temperature-dependent, by a temperature coefficient of
around 0.3% per °C.
•
The pH Meters hold provisions to improve the pH Measures as the
temperature changes and it is termed as Automatic Temperature
Compensation (ATC).
•
The output of the Impedance Voltmeter is Voltage studies and it
possesses to be calibrated to prepare precise pH Measurement.
•
Calibration is performed by immersing the Measuring Electrode into
Buffer Liquid of known pH which assists in understanding millivolt
reading as pH measurement of the Sample Suspension at the
delivered temperature.
Blood Gas Analyzers
Blood gas analyzers are used to measure the pH, partial pressure of
carbon dioxide (pCO2) and partial pressure of oxygen (pO2) of the body
fluids with special reference to the human blood.
The measurements of these parameters are essential to determine the
acid-base balance in the body.
A sudden change in the pH and pCO2 could result in cardiac arrhythmias,
ventricular hypotension and even death.
This shows the importance of the maintenance of physiological neutrality
in blood, and consequently the crucial role that the blood gas analyzers
play in clinical medicine.
Acid Base balance
•
The normal pH of the extracellular fluid lies in the range of 7.35 to
7.45, indicating that the body fluid is slightly alkaline. When the pH
exceeds 7.45, the body is considered to be in a state of alkalosis. A
body pH below 7.35 indicates acidosis. Both acidosis or alkalosis are
disease conditions widely encountered in clinical medicine.
•
Any tendency of the pH of blood to deviate towards these conditions
is dealt with by the following three physiological mechanisms:
(i) buffering by chemical means,
(ii) respiration,
(iii) excretion, into the urine by kidneys.
Blood pH measurement
•
The acidity or alkalinity of a solution depends on its concentration of
hydrogen ions. Increasing the concentration of hydrogen ions makes a
solution more acidic, decreasing the concentration of hydrogen ions
makes it more alkaline.
•
The amount of hydrogen ions generally encountered in solutions of
interest is extremely small and, therefore, the figure is usually
represented in the more convenient system of pH notation. pH is thus a
measure of hydrogen ion concentration, expressed
•
logarithmically. Specifically, it is the negative exponent (log) of the
hydrogen ion concentration.
pH = –log (H+)
Electrodes for Blood pH Measurement:
•
They are all of the glass electrode type but made in different shapes so that
they may accept small quantities of blood and yield accurate results.
•
The most common type is the syringe electrode, which is preferred for the
convenience of taking small samples of blood anaerobically.
•
The small ‘dead space’ between the electrode bulb and the inner surface of
the syringe barrel is usually filled with dilute heparin solution to prevent
blood coagulation.
•
Before making measurements, the syringe should be rolled between the
hands to ensure thorough mixing.
•
Micro-capillary glass electrodes are preferred when it is required to
monitor pH continuously: for example during surgery. These types of
electrodes are especially useful when a very small volume of the sample is
•
Figure shows the constructional details of a typical blood pH electrode
and the measurement set-up used in practice.
Blood PCO2 measurement
•
The blood pCO2 is the partial pressure of carbon dioxide of blood
taken anaerobically. It is expressed in mmHg and is related to the
percentage CO2.
•
Blood gas analyzers make use of a pCO2 electrode. It basically consists
of a pH sensitive glass electrode having a rubber membrane stretched
over it, with a thin layer of water separating the membrane from the
electrode surface.
•
The technique is based on the fact that the dissolved CO2 changes the
pH of an aqueous solution.
•
TheCO2 from the blood sample defuses through the membrane to
form H2CO3, which dissociates into (H+) and (HCO–3) ions.
•
The resultant change in pH is thus a function of the C02 concentration
in the sample.
Construction of pCO2 electrode
Blood PO2 Measurement
•
The partial pressure of oxygen in the blood or plasma indicates the
extent of oxygen exchange between the lungs and the blood, and
normally, the ability of the blood to adequately perfuse the body
tissues with oxygen.
•
The partial pressure of oxygen is usually measured with a polarographic
electrode. There is a characteristic polarizing voltage at which any
element in solution is predominantly reduced and in the case of
oxygen, it is 0.6 to 0.9 V.
•
In this voltage range, it is observed that the current flowing in the
electrochemical cell is proportional to the oxygen concentration in the
solution.
This type of electrode consists of a platinum cathode, a silver/silver chloride
anode in an electrolyte filling solution and a polypropylene membrane.
The electrode is of a single unit construction and contains the reference
electrode also in its assembly.
The entire unit is separated from the solution under measurement by the
polypropylene membrane.
Oxygen from the blood diffuses across the membrane into the electrolyte
filling solution and is reduced at the cathode.
The circuit is completed at the anode, where silver is oxidized, and the
magnitude of the resulting current indicates the partial pressure of oxygen.
Blood cell counters
•
Changes in the normal functioning of an organism are often
accompanied by changes in the blood cell count.
•
Therefore, the determination of the number and size of blood cells
per unit volume often provides valuable information for accurate
diagnosis.
•
The blood constitutes 5–10% of the total body weight and in the
average adult, it amounts to 5–6 l.
•
Blood consists of corpuscles suspended in a fluid called plasma in the
proportion of 45 parts of corpuscles (cells) to 55 parts of plasma.
•
The percentage of cells in the blood is called the haematocrit value or
packed cell volume (PCV).
•
The majority of the corpuscles in blood are red blood cells
(erythrocytes), others being white blood cells (leucocytes) and
Methods of cell counting
Microscopic method
•
The most common and routinely applied method of counting blood
cells even today, particularly in small laboratories, is the microscopic
method in which the diluted sample is visually examined and the cells
counted.
•
Commonly known as the counting chamber technique, it suffers from
several common drawbacks.
•
Apart from the inherent error of the system, which may be about
10%, there is an additional subjective error of ±10% entailing poor
reproducibility of the results.
•
Furthermore, the lengthy procedure involved results in the rapid tiring
of the person making the examination.
•
Automatic optical method
•
The method is based on collecting scattered light from the blood cells
and converting it into electrical pulses for counting. Figure 16.2 shows
one type of arrangement for the rapid counting of red and white cells
using the optical detection system.
•
A sample of dilute blood (1:500 for white cells and 1:50,000 for red
cells) is taken in a glass container.
•
It is drawn through a counting chamber in which the blood stream is
reduced in cross-section by a concentric high velocity liquid sheath.
•
A sample optical system provides a dark field illuminated zone on the
stream and the light scattered in the forward direction is collected on
the cathode of a photomultiplier tube.
•
Pulses are produced in the photomultiplier tube corresponding to
each cell. These signals are amplified in a high input impedance
Optical method of counting cells
Electrical conductivity method
•
Blood cell counters, operating on the principle of conductivity change,
which occurs each time a cell passes through an orifice, are generally
known as Coulter Counters.
•
The technique is extremely useful for determining the number and
size of the particles suspended in an electrically conductive liquid.
•
The underlying principle of the measurement is that blood is a poor
conductor of electricity whereas certain diluents are good conductors.
•
For a cell count, therefore, blood is diluted and the suspension is
drawn through a small orifice.
•
By means of a constant current source, a direct current is maintained
between two electrodes located on either side of the orifice.
•
As a blood cell is carried through the orifice, it displaces some of the
conductive fluid and increases the electrical resistance between the
•
The particles, suspended in a known volume of electrically conductive
liquid, flow through a small aperture having an immersed electrode
on either side; the particle concentration is such that the particles
traverse the aperture substantially one at a time.
•
Each particle passage displaces electrolyte within the aperture,
momentarily changing the resistance between the electrodes and
producing a voltage pulse of magnitude proportional to particle
volume.
•
The resultant series of pulses is electronically amplified, scaled, and
counted.
Errors in electronic counters
•
Aperture Clogging: Partial clogging of the measuring aperture adversely
affects the results of counting. The aperture diameter becomes
constricted and even small particles that were not intended for counting
get included in the final count.
•
Uncertainty of Discriminator Threshold: The uncertainty of triggering at
threshold level and the threshold hysteresis means that particles of the
same size are sometimes counted and sometimes left out.
•
Coincidence Error: Coincidence error will be observed if more than one
particle passes through the measuring aperture simultaneously.
•
Settling Error
•
Statistical Error
•
Error in Sample Volume
•
Error due to Temperature Variation
•
Biological Factors
•
Dilution Errors
•
Error due to External Disturbances
Blood Flow measurements

Look at;
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What is wearable technology
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Its role in healthcare and medical applications
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Any project idea???…simple one???