CCLLIINNIICCAALL CCHHEEMMIISSTTRRYY

Biochemistry is the study of different chemical reactions taking place inside the body. It, therefore,

serves as a tool in providing information on the biochemical basis of a disease.

Our body is made up of biochemicals. These bio chemicals are arranged in a specific order to

perform various processes. Clinical chemistry is the study of the biochemical changes in a human

body. A clinical chemistry laboratory analyses the chemical constituents of various body fluids such

as serum, plasma, urine etc. This reflects the biochemical disorders of various body organs that

further helps in the diagnosis and treatment of the disease.

Terminologies & Definitions

Serum: - Venous blood is preferred for most determinations made on blood. If the blood is collected

in a plain container and allowed to clot, the clot shrinks and the liquid oozes out on standing. On

centrifugation, the blood separates in two layers; liquid as upper layer and a clot at the bottom. The

liquid is called “SERUM”; this does not contain clotting factors.

Plasma: - When blood is collected in a container, having anti coagulant; the blood does not clot but

separates in two layers on standing or on centrifugation. The liquid, as upper layer, is called

“PLASMA” and the cells at the bottom. The plasma contains clotting factors.

Anti coagulant: - The chemicals, which prevent blood from clotting, are called anti coagulant. E.g.

sodium and potassium salts of EDTA (Ethylene Diamine Tetra Acetic Acid), sodium citrate,

Heparin, Fluoride salts etc. The anti coagulants are used depending on the test to be carried out on

the sample.

Distilled water (D/W):- Boiling ordinary water removes dissolved gases, airborne materials, dust,

non-volatile substances etc. there by making it pure and free from all contaminants. Water so

prepared using a special apparatus is called Distilled Water (D/W).

Deionised Water (D.I.):- Deionised water is prepared by passing water through a bed of ion

exchange resins to remove the ions from water. When water passes through the resins, exchange of

ions takes place and ions from water are removed making it ion free.

Enzyme: - Enzymes are biological compounds, which act as catalyst for various chemical reactions

in the body. All these enzymes are proteins and are synthesised by the body. The enzymes are

specific in their activity. A particular enzyme will act on a particular type of substrate. E.g., Glucose

Oxidase will act only on D-Glucose.

/Serum

/Clot

Substrate: - A compound physiologically converted to a product by an Enzyme is referred as its

substrate. Glucose is the substrate for the enzyme Glucose Oxidase.

Reagents: - Chemicals which facilitates the determination and evaluation of clinically relevant

analytes/ constituents of serum or plasma their concentration or activity. The different reagents are

used for different analytes / constituents.

Blank: - This is either distilled/ deionised water or the reagent solution alone. The reagent blank is

required to eliminate the colour of the reagent itself.

Standard: - A reference solution containing known concentration (amount) of the substance under

evaluation 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. Glucose Standard,

Urea Standard etc.

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. The value chart for

each lot is provided.

Control: - It is generally pooled sera having MEAN / TARGET value & SD range for each analyte.

It is analysed for quality control purposes i.e. to evaluate working of system i.e. reagent, instruments

etc.

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.

PRECISION: - The repeatability of a particular value performed “n” number of times. Fig 1

denotes that the results are reproducible but are not near the target.

ACCURACY: - The closeness to the true value. Fig 2 shows that the result is near to the target.

PRECISION AND ACCURACY: The third fig shows that the results are near to the target and are

reproducible.

PRECISION ACCURACY PRECISION AND ACCURACY

Sensitivity: - The ability to detect the lowest possible concentration.

Specificity: - The ability of reagent to react specifically with the substance to be analysed.

Linearity: - The highest possible concentration of the desired substance which can be detected by a

reagent in a specimen without altering procedure. Up to this limit, the linear relation between

absorbance and concentration holds good. Beyond this limit, there is no linear relation between

absorbance and concentration.

Stability: - The period after the reconstitution of reagent, up to which stable reproducible results are

obtained using that reagent.

Incubation: - The period suitable for optimum reaction between the reagent and sample after

addition of sample to the reagent at specified temperature.

Reconstitution:- Addition of fixed volume (as specified in the pack insert/ label) of diluent usually a

buffer or deionised water to the reagent in powder form, so as to convert it into liquid form and make

it ready to use is termed as reconstitution.

Reconstituted Stability: - The time interval after reconstitution and the reagent giving acceptable/

reliable results is its reconstitution stability when stored under specified storage conditions.

Absorbance: - A property of substance to absorb a light this is also referred as optical density i.e.

O.D. The absorbance is proportional to the concentration of substance.

CSF: - Cerebral Spinal Fluid (fluid drawn from lumber region)

Some other Fluids

Synovial Fluid: - Fluid withdrawn from bone joints.

Pleural Fluid: - Fluid withdrawn from pleural cavity.

Amniotic Fluid: - Fluid withdrawn from amniotic cavity (a fluid around the fetus).

Lipemic :- Serum /plasma appears turbid if the lipid concentration (Triglycerides, Cholesterol) in

that sample is high, the phenomenon is known as lipemia & the sample having such turbidity is

known as Lipemic sample. The Lipemic samples may interfere with some of the biochemistry tests.

Haemolysis: - Breaking of RBCs is called haemolysis. If the cell wall of RBCs breaks open, the

haemoglobin enters into the serum/ plasma; turning the fluid reddish in colour. Such sample is called

as Haemolysed sample. Haemolysed samples interfere with most of the biochemistry tests.

Icterus: - Because of the high concentration of bilirubin the serum / plasma is dark yellowish –

reddish in colour. The serum/ plasma are referred as Icteric serum / plasma. Highly Icteric samples

do interfere with biochemistry tests.

PHOTOMETRY Photometry is the science that deals with the measurement of light absorbed by a solution. It helps

in measuring the concentration of any unknown substance.

Basic Principles of Colorimetry

Light is really a form of energy and it moves in space in the form of waves. The distance between

two identical points on a wave cycle is called the wavelength. The unit of measure for wavelength

is nanometre (nm). Wavelength is also called lambda ( ).

Example:

Colours are actually the wavelengths that we see. It is the wavelength that determines the colour of

the light.

The total light spectrum can be divided into 3 distinct regions:

1. Ultraviolet region: Less than 400 nm. It is not visible to the human eye.

2. Visible region: 400 – 700 nm. It is visible to the human eye.

3. Infra red region: more than 700 nm. It is not visible to the human eye.

ULTRAVIOLET INFRARED

300 350 400 450 500 550 600 650 700 750 800 nm

Light Spectrum

VISIBLE

SPECTRUM

Light Source

Filter / Gratting

Reaction Cell

Path Length

Photo detector

A 1/ T, T V

Voltage

When a light falls on to a solution, it either is reflected, absorbed or transmitted through. Photometer

measures transmitted light. The color of a substance will depend on the particular wavelength

absorbed by the substance and the wavelengths, which are transmitted to the observer’s eye.

By measuring what is absorbed, you can measure what is transmitted (without being absorbed).

Thus, we can determine concentration of a biochemical substance from the amount of light

transmitted which is the basis for the diagnosis of a disease.

The Beer – Lambert’s law (or Beer’s law) is the linear relationship between absorbance and

concentration of an absorbing species. It states, “The amount of light absorbed by a solution is

directly proportional to the concentration and path length of the absorbing solution”.

1. Percentage Transmission – It is the amount of light, which passes through a coloured solution

compared to the amount of light, which passes through a blank or colourless solution. As the

concentration of the coloured solution increases the amount of light absorbed increases, while the

%T decreases. Thus the concentration of a substance is inversely proportional to the %T.

2. Optical Density – The O.D. is more preferred in clinical chemistry since it has a direct

relationship with the concentration of a solution, i.e. as the concentration of a solution increases,

the absorbance or O.D. also increases.

Following formula is used to convert %Transmission (%T) into O.D.

The basic components of all photometers and spectrophotometers are as follows:

Light Source:

Halogen Tungsten lamp is generally used as the light source. A regular voltage supply is very

important for a stable source since fluctuations of voltage is a serious problem to a photometer

system.

O.D. = 2 - log T

Wavelength Selectors

In most instruments filters are used for this purpose, but in the more expensive type of

equipment a prism or diffraction grating is used to obtain the required wavelength. In case of

prism or diffraction grating the precise wavelength selection (± 1 nm) is possible whereas in

case of filters what we get is band of wavelength (± 8 – 10 nm)

The filters chosen are usually complementary to the colour of the solution to be measured

(see table below).

Colour of Solution Complementary Filter

Blue Yellow

Bluish - green Red

Purple Green

Red Bluish-green

Yellow Blue

Yellowish - green Violet

Cuvettes and Flow-through cells

These are used to hold solutions. The cuvettes can me made up of glass or of polystyrene.

Flow cells are generally of steel body with quartz windows.

Photo detector

Light falling on these elements generates an electric current, which is proportional to the light

intensity falling over it.

Clinical chemistry automation is the mechanization of the steps in a procedure.

The steps of biochemical analysis can be broadly divided as:

1. Sample handling

2. Reagent handling

3. Sample processing

4. Analytical procedures e.g. mixing, heating etc, reaction analysis, reading the absorbance and data

storage.

SEMI- AUTOMATED ANALYZER

In the semi automated analyzer pipetting, mixing and incubation etc. are done by the

Technician. Solution is aspirated into the Instrument, it measures the O.D., calculates the

Concentration and prints / stores the data for subsequent use.

The advantages of these instruments are that the conditions of the test can be programmed and

results can be saved. The working can be faster and manual error in computing are minimised.

These instruments are highly effective in measuring enzyme kinetics.

FULLY AUTOMATED ANALYZERS

In fully automated analyzer pipetting and mixing of reagent and sample, incubation, measuring the

O.D.’s, calculations and finally printing of the results is done by the instrument itself.

A fully automated analyzer enables the lab to:

Better utilize its resources by giving higher throughput (number of tests performed in one hour).

Enhanced productivity.

Removes chances of manual errors & enables faster sample turnaround.

Requires less volume of reagent therefore is more economical.

BIOCHEMISTRY PRINCIPLES

End-Point

The sample and the reagent mixture (Reaction Mixture) are incubated at specific temperature for the

specified time. The colour development takes place. The absorbance of the mixture is measured at

the end of the incubation period at a specific wavelength. This absorbance remains constant for some

time as the reaction between sample and reagent has been completed and there is no further change

in the absorbance. These are called as end point reactions.

E.g. Glucose, Triglycerides etc.

For some tests “Factor” is provided by reagent manufacturer e.g. Total & Direct bilirubin.

END - POINT CALCULATION OF RESULTS

Sample Conc. = STD Conc. X (Sample O.D - RB O.D.)

STD O.D – Reagent Blank O.D.

Result = Factor X (Sample O.D- RB O.D.)

Factor = STD Concentration

STD O.D. - Reagent Blank O.D.

Initial Rate

Other names- Fixed Time, Fixed point, Two point kinetic.

In this measurement mode after the preparation of sample and reagent mixture some time is allowed

before taking the first reading. This is called as lag phase or delay time. This delay or lag phase is

required for the reaction to stabilize. After the completion of the delay time, first O.D. measurement

is taken. After certain time interval the second O. D. reading is taken. The second reading is taken

during the linear phase of the reaction. Samples and standard are performed in same manner. The

two readings are taken at the fixed time intervals and change in absorbance (∆ O. D.) is calculated

for the fixed time interval. The results are calculated as per the following equation...

E.g. Urea – GLDH, Creatinine.

Initial Rate

CALCULATION OF RESULTS

Sample Conc = STD Conc

X ∆ A Sample

∆ A STD

Factor =

Standard Concentration

A STD

Result = Factor X A Sample

Ab

sorp

tio

n O

D

Time T T1

A1 A

2

T

2 T1

∆O.D

.

T

Increasing

A2

A1

Decreasing

T

2 T1

O

.D.

T

KINETIC

In this measurement mode, the absorbance readings are taken when the reaction is in progress. After

mixing of the sample and reagent, some time is allowed to stabilize the reaction. This is called lag

phase or delay time. After completion of the Lag phase/Delay time the absorbance readings are

readings taken over a period of time in the linear phase of the reaction and average Change in

absorbance per minute (∆ O. D. / min) is calculated. ∆ O. D. / min are used to calculate the enzyme

activity as per the following formula.

Kinetic

CALCULATION OF RESULTS

Result = Factor X Mean ∆ O. D. / min

The kinetic reactions can be of two types:

Increasing: - Alkaline Phosphatase, Amylase, Creatinine Kinase, GGT etc.

Decreasing: - SGOT (AST), SGPT (ALT)

Bichromatic:

In this mode, the absorbance readings are taken at two different wavelengths of the same solution.

One at primary wavelength (where the colour is developed because of the reaction between sample

and reagent is measured) and other reading is taken at secondary wavelength (Where the absorbance

of the interfering substances present in the sample is measured). The difference between the two

absorbance readings is taken in to consideration for calculating the result. This helps to minimize the

interference of interfering substances (Icteric, Lipemic and Haemolysed.

Sample Conc. = STD Conc. X (Sample O.D 1- Sample O.D 2.)

(STD O.D 1 – STD O. D. 2)

Decreasing

O.D.1

O.D.2

O.D.3

1 MIN 2 MIN 4 MIN 3 MIN

A4

A

3 A

2

A1

A1

A

2

A

3

A4

Increasing

g

1 MIN

O.D.3

O.D.2

O.D.1

2 MIN 4 MIN 3 MIN

Differential Blanking (Sample Blanking)

Two reaction mixtures are prepared. In one, the actual reaction between the sample and reagent takes

place and in other the colour development is only due to the colour of the sample itself. The

difference between two absorbances is used to calculate the final result.

This helps to eliminate the sample colour interference.

Multi Point Calibration

This is also referred as Non-linear Calibration .These are generally end point chemistries (1 – point

non-linear). The graph of absorbance against the concentration is plotted. The concentration of the

sample is derived from the graph using the absorbance of the unknown.

The examples are RA, CRP, ASO.

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

0 100 200 300 400 500 600

0.000

0.500

1.000

1.500

2.000

2.500

0 500 1000 1500 2000 2500 3000 3500