Practical Clinical

Biochemistry
Introduction

• It deals with the applications of biochemistry

laboratory to find out the cause of a disease.
• Clinical chemistry measures levels of chemical
components in body fluids. These chemicals are
called biochemicals.
• Biochemicals measured may include blood
glucose, electrolytes, enzymes, hormones, lipids
(fats), other metabolic substances, and proteins.
• The results of these measurements is quantitative,
meaning there will be numbers involved stating
“how much.”
 Spectroscopy and Clinical Biochemistry

Many assays in biochemistry depend on measuring the light

absorbed by a substance in the visible or ultraviolet region of
the electromagnetic spectrum. The estimation is based on
comparison of the light absorbed by substances in solution of
unknown concentration with the light absorbed by the same
substance in solution of known concentration a good estimate
of sample concentration can be made. This, at its simplest, is
spectroscopy (or spectrophotometry,. This method can be
used in estimation of coloured substance; and substances,
which absorb only in the UV, such as protein and nucleotides;
and colourless substances such as sugars, which can undergo
a series of chemical reactions to give a coloured derivative.
 
Characteristics of wavelength

Light and all electromagnetic radiation consist of

photons or packets of energy traveling in waves.
The electromagnetic spectrum is described in term
of wavelength (λ) which is the distance between
successive wave peaks and its unit is nanometer
(nm) or A.
Principles of Spectroscopy
Deals with the production, measurement, and interpretation
of spectra arising from the interaction of electromagnetic
radiation with substance.

Electric Spectrum of Energy

The gamma rays (wavelength < 0.1 nanometres) to radio
rays (wavelength > 250 millimetres).

Spectroscopy Deals With

The ultraviolet (180 to 380 nanometres), the visible (380 to
800 nanometres), and the infrared (0.8 to 50 micrometres).
Electromagnetic Spectrum of Energy
The gamma rays (wavelength < 0.1 nanometres) to radio rays
(wavelength > 250 millimetres).
Absorbance Measurement for
Ultraviolet or Visible Light
Absorbance is measured in a spectrophotometer,
which provides a light source with selectable
wavelength in the range 180 to 800 nm
(ultraviolet and visible light).
Sample is contained in a cuvett of exactly 1.00
cm thickness.
The light passing through the sample is then
measured and recorded by a detector.
Spectrophotometer
Spectrophotometer

• It's an instrument used to measure light that is

either absorbed or transmitted through a solution
in cuvette.
• Transmitted light is internally mathematical
converted into absorbance unit to determine
[light absorbing substance] present in the cuvette.
• All spectrophotometers have basically the same
components including light source,
monochromator, sample holder, detector and read
out device.
Light Source

• A suitable light source must meet the following

requirements:
1. It must produce a beam with sufficient power,
2. It must provide a continuum of wavelength over the
region of interest,
3. It must stable.
• A tungsten lamp provides a continuum of wavelength, and
it's not used for measurements below 350 nm.
• Hydrogen and deuterium discharge lamp give a
continuous emission spectrum in the ultraviolet (UV)
region, which ranges from 195 – 380 nm.
• Mercury, xenon, and other types of lamps are available.
Entrance and Exit Slits
Monochromator:
• It's used for isolation of required range of wavelength; this can be
done by using of filters, prisms, and differentiation gratings & are of
2 types:
1. Absorption filters; produce a wide range of wavelengths.
2. Interference filters; are made at fixed wavelengths.

Sample Holder:
• It's also known as cuvette or cell; made of glass, quartz or plastic.
1. Glass and transparent plastic cuvettes are used in the visible region.
2. Quartz or silica cell are necessary for UV radiation.
• Cuvettes are of two types – macrocuvettes with a capacity of 3 ml
only and microcuvettes with a capacity of 1 ml only.
Detector:
It converts the electromagnetic radiation transmitted by a
solution into an electrical signal. Four types of detectors
can be used to measure the transmitted light:
1. Barrier layer cell
2. Silicon photodiode
3. Phototube
4. Photomultiplier
 
Readout Device:
The magnitude of the electrical current from a detector
can be displayed on a meter; digital readout device or
recorder.
Components of a Spectrophotometer
Characteristics of Wavelength

• Light and all electromagnetic radiation consist of

photons or packets of energy traveling in waves.
• The electromagnetic spectrum is described in
term of wavelength (λ) which is the distance
between successive wave peaks and its unit is
nanometer (nm) or A.
The Electromagnetic Spectrum

Ultraviolet invisible light (< 380 nm) Violet visible light (380 to 440 nm)
Blue visible light (440 to 500 nm) Green visible light (500 to 580 nm)
Yellow visible light (580 to 600 nm) Orange visible light (600 to 620 nm)
Red visible light (620 to 750 nm) IR Invisible light (750 to 2000 nm)
Laws of Absorption of Light

• There are two common methods of expressing the amount of light

absorbed by a solution.
• [Link] % transmittance.
• 2. By optical Density (O.D) Absorbancy (A) or Extinction (E) of
the solution.
• Absorbance, transmittance, and Beer’s law:
Beer-Lambert’ law

When a ray of monochromatic light of initial

intensity Io passes through a solution in a
transparent vessel, some of the light is absorbed so
that the intensity of the transmitted light I is less
than Io. There is some loss of light intensity from
scattering by particles in the solution and reflection
at the interfaces, but mainly from absorption by the
solution. The relationship between I and Io depends
on the path length of the absorbing medium, I, and
the concentration of the absorbing solution, c. these
factors are related in the laws of Lambert and Beer.
• Absorbance can’t be measured directly but
mathematically derived from % T. According to
Beer-Lambert law
• Where,
A: Absorbance
a: absorptivity constant for the substance,
b: length of the light path through the substance,
c: concentration of the substance
Principle of Spectrophotometry
 Determination of Absorpition Maxima

For conc/A. relation to be linear the wavelength chosen to

measure the coloured solution should be that, at which,
light is maximally absorbed. This is determined by using
fixed concentration of coloured solution and measuring it
at different wave lengths. Plot the graph taking A. on Y
axis and wavelength on X axis & show results Graphically
Types of Specimens used in Clinical Chemistry

Tests in clinical chemistry are performed primarily on serum, plasma,

urine and other body fluids.
– Serum is the yellow watery part of blood that is left after blood
has clotted and all blood cells have been removed.
– Plasma is in essence the same as serum, but is in blood which
hasn’t clotting. Plasma is obtained by
centrifugation before clotting occurs.
– Both look similar and can easily be visually confused.
Procedure of Serum Preparation

• Draw blood from patient. Select vacutainer with no

anticoagulant.
• Allow to stand for 20-30min for clot formation.
• Centrifuge the sample to speed separation and affect
a greater packing of cells. Clot and cells will
separate from clean serum and settle to the bottom
of the vessel.
• The supernatant is the serum which can be now
collected by Dropper or pipette for testing purposes
or stored (-20°C to -80°C) for subsequent analysis
or use.
Thank You Very Much
For Paying Attention