CLINICAL CHEMISTRY

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.

/Serum

/Clot

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.

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

VISIBLE SPECTRUM

INFRARED

300

350

400

450

500

550

600

650

700

750

800 nm

Light Spectrum

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 observers 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 Lamberts law (or Beers 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.

O.D. = 2 - log T
The basic components of all photometers and spectrophotometers are as follows:

Light Source Reaction Cell

Photo detector

Voltage

Path Length

A 1/ T, T V Filter / Gratting

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.

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 Blue Bluish - green Purple Red Yellow Yellowish - green Complementary Filter Yellow Red Green Bluish-green Blue 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. 2. 3. 4. Sample handling Reagent handling Sample processing 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
X (Sample O.D - RB O.D.)

Sample Conc. =

STD Conc.

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.

Absorption O D

Time T Initial Rate

T1

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...
Decreasing O.D.

A2 O.D.

A1

T1

T 2

A1

A2

Increasing

T1

T 2

E.g. Urea GLDH, Creatinine. Initial Rate CALCULATION OF RESULTS Sample Conc =
STD Conc A STD X A Sample

Factor =

Standard Concentration A STD

Result = Factor X A Sample

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
Increasing

g A1 A2 A3 A4
O.D.3 O.D.2 O.D.1

Decreasing A4 A3 A2 A1
O.D.1 O.D.2 O.D.3

1 MIN 2 MIN 1 MIN 2 MIN 3 MIN 4 MIN

3 MIN

4 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)

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.

2.500
1.600 1.400 1.200 1.000 0.800

2.000

1.500

1.000
0.600 0.400 0.200 0.000 0 100 200 300 400 500 600

0.500

0.000 0 500 1000 1500 2000 2500 3000 3500