 Biochemistry can be defined as the study of the chemical

processes and reactions that take place within living

organisms.

 Clinical Biochemistry is the area of pathology that is

generally concerned with the analysis of body fluids for
diagnosis of any pathogenesis
.
BIOCHEMISTRY

The body of a healthy human contains definite

concentrations of different Biochemical Molecules.
Extensive studies have helped to determine the
Normal Concentrations of each such biochemical
molecule.
But when the chemistry of the cell is altered, the normal process
breaks down triggering elevated levels of biochemical
molecules or unusual chemicals to be synthesized. These
chemicals find their way out of the cell and into various body
fluids.

By estimating these chemicals in various body fluids one can

deduce the clinical significance and subsequent prophylaxis.
TERMINOLOGIES & DEFINITIONS

Whole blood: Properly mixed blood is collected in anti coagulant in container.

Serum: When blood is withdrawn and allowed to clot

in a container (usually a tube); on centrifugation, the
blood separates into two layers; liquid as upper layer
and a clot at the bottom. The liquid is called “SERUM”;
which does not contain clotting factors.

Plasma: When blood is collected in a container,

having anti coagulant; the blood does not clot but
separates into two layers on standing or centrifugation.
The liquid, as upper layer, is called “PLASMA” and the
cells at the bottom. The plasma contains clotting
factors.
 TERMINOLOGIES & DEFINITIONS

Anti coagulant: The chemicals which prevent clotting of blood are called anti
coagulant. E.g. EDTA, Heparin, Fluoride salts etc.

Standard: A reference solution containing known concentration of the substance

is called “STANDARD”. This is primary reference material, with which the test
sample is compared in order to determine the concentration of analyte in that test
sample.

Calibrator: A serum based standard having established values for many analytes
(tests) is known as “CALIBRATOR”. This is a secondary reference material, with
which the test sample is compared in order to determine the concentration of
analytes in that test sample.

Control: It is generally pooled sera having MEAN / TARGET value & SD range for
each Analyte. It is analyzed for quality control purposes i.e. to evaluate working of
system i.e. reagent, instruments and techniques etc.
 TERMINOLOGIES & DEFINITIONS

Enzyme:
Enzymes are biological compounds which act as catalyst (accelerate the rate of reaction) for
various reactions in the body. They act with specific substrate.
Substrate:
A compound ,that is getting physiologically converted to a product by an Enzyme is referred
as it’s substrate.
S P
Sensitivity:
The ability of the reagent to detect the lowest possible concentration of analyte in the
sample.
Specificity:
The ability of reagent to react specifically with the substance, intended to be analyzed.
Linearity:
The highest possible concentration of the desired substance which can be detected by a
reagent in a specimen without altering procedure(dilution of sample).
TERMINOLOGIES & DEFINITIONS

Stability: The period after the reconstitution of reagent, up to which the

reagent is able to recover the control values in reproducible manner.
Absorbance: A property of substance to absorb light this is also referred to as
optical density i.e. O.D. The absorbance is proportional to the concentration of
substance.
CSF: Cerebrospinal Fluid, This is the fluid around cerebrospinal system( fluid
drawn by Lumbar Puncture)

Some other Fluids:

Synovial Fluid: Fluid withdrawn from Synovial bone Joints.
Pleural Fluid: Fluid withdrawn from Pleural Cavity.
Amniotic Fluid: Fluid withdrawn from Amniotic Cavity (a fluid around the
fetus).
 TERMINOLOGIES & DEFINITIONS

L.I.H.
Lipeamic: Serum /plasma appears turbid if the lipid concentration in that sample is
high, the phenomenon is known as lipemia & the sample having such turbidity is
known as lipemic sample.

Icterus: When serum / plasma is more yellow than usual, the yellowness is called
Icterus. The serum/ plasma is referred as Icteric serum / plasma.

Haemolysis: Breakdown of RBCs is called Haemolysis. If RBCs break open, the

haemoglobin comes out into the serum/ plasma; such sample is called as
haemolysed sample.

Note: these three states of serum are not fit to be used as sample.
 TERMINOLOGIES & DEFINITIONS

Mean / Target:
The expected value for the particular analyte, which is mentioned in the value
sheet. It is method dependent. Some products available in market; provide
different values for different methods as well as for different analyzers for same
chemistry.

SD Standard Deviation:
It is the deviation allowed from the target value of the assayed values of the
control, which is determined by the manufacturer and mentioned in value sheet.
 TERMINOLOGIES & DEFINITIONS

Accuracy
Accuracy is agreement between run value & target value. i.e.
closeness of obtained value from target value is Accuracy.

Precision
Precision is agreement between value obtained in repeated runs. i.e.
Closeness in values of repeated run is precision.
TERMINOLOGIES & DEFINITIONS

ACCURACY & PRECISION

TERMINOLOGIES & DEFINITIONS

ACCURACY & PRECISION

Photometry

Beer & Lambert’s Law

Biochemistry Principles
LIGHTS AND WAVELENGTHS

When a beam of white light passes through a prism, it splits into its seven basic
colours such as violet, indigo, blue, green yellow, orange and red.
LIGHTS AND WAVELENGTHS
 Colorimeter / spectrophotometer

If we allow a beam of light to fall on a coloured solution, it will absorb all colours
other than red. Hence the solution appears red in colour.

Incident Light
lo lT

Transmitted Light

lA
Absorbed Light
BEER’S & LAMBERT’S LAW

c
A
A = Ebc

Where,
A is Absorbance
E is Molar Absorbtivity
b is path length of sample / path length of cuvette in cms
c is concentration of compound in solution

I0- Incident
If E and b are constant Light
C–
Aαc
Concentration
α – Light Path
I1 – Emerged
light
 Optical Density

LIGHT SOURCE CUVETTE CONTAINING RED SOLUTION COMPLEMENTARY GREEN PHOTO DETECTOR
FILTER

 When the white light passes through a coloured solution in the Cuvette, all its
components are absorbed except for a particular wavelength corresponding
to the colour of the solution; which is transmitted.

 When this light passes through the Filter, only a specific wavelength
(complimentary to the coloured solution) is allowed to pass; which is then detected
by the Photo Detector.
 Components of Photometers

Light Source

Different source is necessary depending on the

wavelength and intensity of light required

Tungsten Halogen Lamp

For Visible & UV Region
Wavelength Selectors

Filters all other wavelengths and allows to pass light of only max
wavelength

INTERFERENCE FILTERS
 Sophisticated types of filters.
 It consists of successive layers of dielectric material of controlled
thickness between two thinly silvered pieces of glass.
 More narrow band path.
 Bandwidth is  8 to 15 nm.
 This is the most preferred type of filter used in most semiautos & some
FAAs.
Wavelength Selectors

PRISMS – True Prism &

Grating

 Separates white light into a

continuous spectrum by refraction.
 For visible region glass prisms, for
ultraviolet region on quartz or
silica prisms.
 Spectral purity is greater than
filter.
 Bandwidth is less than
interference filters (0.5 nm).
 More expensive.
 Affected by temperature &
Moisture .
 Example of grating – EM 360/XL
640
 Reaction Cell

CUVETTE & FLOW CELL

Used to hold colored or reaction solution, also

called as cell.

 For use between 320 to 950 nm - Glass Cuvettes are satisfactory.

 For measurement below 320 - quartz cell is required.

It may be round, or rectangular in shape.

Photo Detectors& Read Out Devices

A Photo detector converts and amplifies the light energy to an

electrical current.

Sample
Meter

The current from the detector is fed to a sensitive suitable readout device. It

might be scalar-like galvanometer or digital display i.e. LED (Light Emitting

Diodes).
Quick run through ……………..

What is biochemistry?

Components of Photometer

Beer and Lambert’s Law

 WHAT IS AUTOMATION ?

Automation is the mechanization

of steps in a procedure.

In a clinical laboratory, it is a self regulating process, wherein the

specimen is accurately pipetted by a mechanical probe in instrument
and mixed with a predetermined volume of the reagent in a Cuvette
and final results are displayed in digital forms and also print formats.
WHAT IS AUTOMATION ?

Reagent Pipetting

Manual Sample Pipetting

Fully Auto
Incubation
Analyzer

Measuring
Absorbance
Semi auto
Analyzer
Calculating Final
Result
Advantages of using Auto Analyzer

 Minimal Manpower

 Decreased Manual Error

 Minimal Reagent requirement

 Quick Processing

 Economical

 Complete Profile Data storage facility for patient results

 Increased linearity
Advantages of using Auto Analyzer

 Sophisticated built-in Q.C. program

 High accuracy and precision due to;

 In built re run facility

 Accurate pipetting

 Temperature control of sample holder

INSTRUMENT CLASSIFICATION

On degree of Automation, the instruments are classified as:

Manual

Semi Automated

Fully Automated
 INSTRUMENT CLASSIFICATION

On type of Reagents used, instruments are classified into

Closed system : One has to use the reagents specified by the

manufacturer. E.g. Hematology analyzers, Electrolyte

Analyzers, Blood Gas Analyzers, Biochemistry Analyzers

Open system : One can use any brand of compatible

reagents available

for that purpose. E.g. Biochemistry analyzers, Elisa

readers.
 INSTRUMENT CLASSIFICATION

FUNCTIONAL CLASSIFICATION

BATCH ANALYZERS

Batch Analyzers are fully automated analyzers. But these analyzers can
be programmed to perform only one type of a test at a time.

The operator has to reprogramme the analyzer for a different test.

Example “Smart Lab”.
 INSTRUMENT CLASSIFICATION

FUNCTIONAL CLASSIFICATION

RANDOM ACCESS

Random access analyzers, can be programmed to analyze several

Serum Samples for various parameters simultaneously. The operator
has total freedom to assign all or some of the tests to all or some of the
samples.

In short it is a “Complete Walk Away System”

 Quick run through ……………..

Advantages of automation?

Types of instrument based on degree of automation?

Types of instrument based on types of reagents used?

Batch analyzer is similar to random access analyzer?

End Point Assay
1) Incubation at specific
temperature for
specific time duration.
Read Result
2) Reaction ‘ends’/(completes)
after a ‘point’ of time’. Color
develops due to

Absorbance
reaction.
Colour
3) The absorbance of reaction
Stability
mixture i.e.
Color is measured at
specific
wavelength at the End of Incubation
the reaction. Time

4) Reaction direction is Time

mostly increasing.

5) Mostly incubation is
outside the instrument.
 End Point Assay

Test set-up

6) Standard is provided for calibration.

Reagent Reagent Standard
Reagent Blank STD 7) Concentration of Sample
is calculated:

Conc of S = OD(S) – OD(RB) x Std Conc

OD(Std) – OD(RB)

wherein Std Conc is Factor K

Reagent Sample OD(Std) – OD(RB)
Test
8) Measurement is based on the color
development.
e.g.. Glucose, Cholesterol, Total Protein
, Albumin.
 Kinetic or Continuous Rate Assay

1) Multiple (continuous) Abs measurement of reaction

at fixed time interval during Read time

2)  A / min is measured.
Read Time

Absorbance
3) Reaction time is short

4) Reaction Direction is increasing or 4

decreasing Lag
3
Time 2
5) Incubation is inside the instrument 1
6) Measurement starts after prerequisite
interval time (lag time)
Time
 Kinetic or Continuous Rate Assay

Test set-up 7) Standard is not provided.

Distilled 8) Factor is directly provided by the manufacturer and

depends
Water
on molecular absorbtivity of substrate or end product.

Water Blank Conc. Of Sample =  A / min x Factor K

Factor Calculation = Reac Vol x 103

Sample Vol x Absorptivity x Light Path

Reagent Sample
9) Semi Automated Analyzer is compulsory

10) Measurement is based on rate of the light absorbing

capacity of different substrate at different wavelength
Test

eg. SGOT , SGPT, ALP , LDH, CK.

 Initial Rate / Fixed Time
Two Point kinetic assay

1) Multiple (dual) Absorbance measurement

of reaction at fixed time during Read time.
T2
2) Difference between two absolute
measured absorbance at definite
time interval T1

Absorbance
3) Reaction time is normally between
E.P.& Kinetic.
Lag
4) Reaction Direction are increasing or Time Read Time
decreasing

5) Incubation is inside the instrument

Time
6) Measurement start after leaving some
time (lag time)
 Initial Rate / Fixed Time
Two Point kinetic assay

Test set-up
Distilled Reagent STD7) Standard is provided for calibration.
Water
8) Change in Absorbance  A = T2 – T1
Reagent

Water Blank
Conc. Of Sample =  A X Std Conc
STD
A)

Where in Std Conc is Factor K

Reagent Sample A
Reagent

e.g.. Urea, Creatinine

Test
 Bichromatic

• The reaction mixture absorbencies are measured

at Two filters (wavelengths) for same test.

• First filter is Primary filter and 2nd filter is reference

filter. ABS reading of 2nd filter is subtracted from
ABS reading of primary / main filter.

• This helps to take care of interfering substances

like L.I.H. (Lipemic,Icteric & Haemolysed samples).

RESULT:- STDCX Sample ABS1-Sample ABS2

STD ABS1-STD ABS2
 Multi Point Calibration

This is also referred as Non-linear Calibration .

This is generally an End Point chemistry.

A graph of absorbance against the known Standard concentration

is plotted.

The concentration (value) is derived from the plot (graph) using the
absorbance of the unknown.
 Multi Point Calibration

2.500

2.000

1.500

1.000

0.500

0.000
0 500 1000 1500 2000 2500 3000 3500
 Multi Point Calibration

1.600

1.400

1.200

1.000

0.800

0.600

0.400

0.200

0.000
0 100 200 300 400 500 600
 Quick run through ……………..

Types of Assay?

Difference between Kinetic and initial rate assay?

THANK YOU