INTRODUCTION TO BIOCHEMICAL ANALYSIS

Lecture 3 Basic Chemical Pathology MPAK 1112

Brief history of development:

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Mid 19th century - HCl in gastric juice, urine tests for sugar, protein and bile. 1920s - venepuncture routine practice, colorimeters widely available, blood analyses from wards and side rooms to hospital laboratories. 1940 and 1950s - advancement of instrumentation and development of microand ultra-micro methods ===> steep increase in variety of and demand for chemical pathology investigations. 1960 and 1970s - new tests + automatic equipment capable of multiple analysis, e.g. 240 samples per hour, 30 tests per sample ===> computerized data processing ===> screening and profile tests. 1980s - consideration of finance (escalating cost), efficacy and priority (allocation of resources: one patient over-investigated may have to mean another underinvestigated) ===> control of unrestricted demands for analysis, improvement on SELECTIVITY in the choice and frequency of investigations. 1990s - trend in 1980s continues with multidisciplinary laboratory automated systems. further cost-containment;
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DIAGNOSTIC TESTS: Basic Principles, Use and Interpretation

Diagnostic tests
Assists the clinician
1. Screening


to identify risk factors for disease



early intervention to prevent disease occurrence

to detect occult disease in asymptomatic persons.



may reduce disease morbidity and mortality through early treatment.

2. Diagnostic  to help establish or exclude the presence of disease in symptomatic persons.

 

Early diagnosis after onset of symptoms and signs Differential diagnosis of various possible diseases
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3. Patient management


Tests can help to:

(1) evaluate the severity of disease, (2) estimate prognosis, (3) monitor the course of disease (progression, stability, or resolution), (4) detect disease recurrence, (5) select drugs and adjust dosages, (6) select and adjust therapy.

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What is biochemical testing?



Biochemical testing looks at the levels of specific substances and enzymes that are produced by chemical reactions in the body. Metabolic disorders are often the result of a missing or non-functional enzyme.  When an enzyme is missing, greatly reduced in quantity, or is not working properly, the body cannot perform its usual chemical reactions.  As a result, several harmful changes in body biochemistry can occur such as accumulation of substrate, accumulation of precursors, deficiency of products, redirection of substrate into alternate pathways.
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What is biochemical testing?



Biochemical testing looks at the levels of these disturbances and can be used to detect several different metabolic conditions. The analysis of these constituents can be either qualitative or quantitative

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Qualitative analysis


Detection of substances in a mixture by chemical means Intensity of the reaction as shown by colour reaction indicates the amount of substance present

Quantitative analysis


Exact quantity of a substance is measured

 

Volumetric analysis e.g: titration Colorimetric analysis based on chemical reactions that produce coloured substances and detection is by a spectrophotometer Flame emission spectrometry a substance is forced under pressure into a flame which then emits light tant can be measured by a photosensitive detector
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Laboratory tests
A laboratory test:  a procedure in which a sample of blood, urine or other tissues or substances in the body is checked.


often part of the routine examination to detect possible changes in an individuals health before any signs and symptoms appear.

play an important role in diagnosis



Symptoms present

to plan treatment, monitor and evaluate the response to treatment, or monitor the course of illness over a period of time.
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Blood tests


The common blood tests include

       

full blood count, blood chemistry, blood glucose, blood cholesterol, liver function, kidney function, tests for certain infections, blood-clotting studies, blood hormone levels and blood grouping.

Preparation of patient before certain tests:



Eg: instruction not to eat or drink for a certain number of hours before blood glucose estimation.

sample of blood taken from the inside of the elbow or the back of the wrist with a needle.
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Containers for samples

  

Serum plain bottles/tubes Plasma heparinised tubes/containers with EDTA Urine preservatives eg. Glacial acetic acid

Samples


Some blood tests require samples taken over a period of time e.g. blood glucose. Some blood tests require a sample from an artery e.g. estimation of arterial oxygen levels. Such tests are usually carried out in hospitals. If a small blood sample is required e.g. blood glucose estimations, a few drops of blood can be squeezed out from a tiny prick on the fingertip or ear lobe.
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Samples for examinations in a biochemistry laboratory



Serum
 

Plasma
 

the fluid remaining after blood has clotted and the clot has been removed. the clot - the red blood cells, white blood cells, and platelets the fluid which remains after the red cells have been removed from blood clotting has been prevented by use of anticoagulant

  

Urine
 

Faeces


Appearance, SG, microscopy Gastrointestinal blood,

Cerebrospinal fluid (CSF)

the fluid made in the choroid plexus of the ventricles in the brain and secreted into the subarachnoid space around the brain and spinal chord. Specimens for lab examinations are obtained from lumbar puncture
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Collection of specimens and some General Techniques

Blood  Capillary or venous blood is used for almost all determinations made on blood  Capillary blood


Finger or thumb
 

 

Clean with 70% methylated spirit, prick with sharp needle or sterile blood lancets Apply gentle pressure and let blood flow into suitable clean, dry pipette (do not exert undue pressure on the finger/thumb as this will alter some constituents) Collect volumes up to about 200 QL Alternative containers: capillary tubes

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Collection of specimens and some General Techniques

Blood  Venous blood not homogeneous but reflects the anatomy, physiology and biochemistry of tissues


apply torniquet just above the elbow, sterilise the skin, insert disposable sterile needle of disposable syringe into the vein, withdraw plunger slightly, remove torniquet and let blood flow into the syringe barrel. Withdraw needle, remove needle and transfer blood into a container (plain/heparinised)

Arterial blood eg for blood gases Equipment used to collect and transport specimens to the lab
 

Swabs, needles, syringes, Containers

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Storage of specimens


Interval between collection and analysis

  

haemolysis plasma K if blood is left unseparated for a long time some enzymes deteriorate rapidly in vitro due to:  Temperature  Light

Condition of storage
 

 

stability by temperature Alternate freezing and thawing damaging to proteins UV/daylight eg destroys bilirubin Preservatives to prevent bacterial growth esp in urine samples
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Special Considerations for Biochemical Testing Technical considerations  Labelling specimens  Label accurately information should correspond with that in the request form  Blood - Name, date, time, test requested  Urine time & date of starting and completing the collection (24-hr urine)  Faeces time, date,


Record = Reference

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General Principles of Chemical Reactions



Atomic structure
  

Nucleus: protons (+ve) and neutrons Electrons (-ve) surround nucleus Chemical properties controlled by electrons

Atomic numbers and atomic masses of elements



Atomic number: number of protons in the nuclei of atoms Atomic mass/weight: sum of the number of protons and neutrons

Isotopes


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Atoms of the same element that have the same number of protons but different number of neutrons (same atomic number but different atomic mass)

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General Principles of Chemical Reactions



Molecules and molecular weights



Smallest particle of an element or compound that can exist separately

Oxidation and reduction reactions

 

Oxidation: Loss of electrons or hydrogen Reduction: Gain of electrons or hydrogen

LEO GER - lose electrons oxidation, gain electron reduction OIL RIG - oxidation involves loss, reduction involves gain

Electrolytes and electrolysis



Electrolytes: acids, bases or salts which when dissolved in water conduct electric current Electrolytic system: anode and cathode
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General Principles of Biochemical Analyses



Every manipulation in biochemical analysis is a potential source of error so that in the interest of accuracy and precision the number of manipulations be kept to a minimum. There are 6 basic operations in each biochemical analysis:
1. A measured aliquot of the sample is deproteinised 2. A measured volume of the protein free filtrate or supernatant is transferred to the reaction tube 3. Measured volumes of the reagents are then added 4. Samples and reagents are thoroughly mixed 5. The reaction is allowed to proceed for a specific time 6. A product of the reaction i.e colour, fluorescence or electrical signal is measured for the sample, standards and blank.

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General Principles of Biochemical Analyses



Possible worst causes of error:



Measurement of volumes of sample and reagents Timing of the reaction

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Analytical Techniques


    

Photometry  Colorimetry  Spectrophotometry Flame Photometry Electrochemistry Turbidimetry Electrophoresis Immunochemistry

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Principle of Colorimetric Analysis



Colorimetry  Compares the colour produced by an unknown quantity of a substance with the colour produced by a standard containing a known quantity of that substance.  Basis of the principle of colorometric analysis:
 

Many substances are coloured in solution Intensity of colour in these solutions is related to the amount of substance in solution
 

Low concentration pale colour High concentration darker colour

The coloured solutions can absorb light at given wavelengths in the visible spectrum.
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Origin & nature of light energy

Light energy  Source electron activity
e


The ground state of an electron, the energy level it normally occupies, is the state of lowest energy for that electron There is also a maximum energy that each electron can have and still be part of its atom. Beyond that energy, the electron is no longer bound to the nucleus of the atom and it is considered to be ionized.

orbit

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When an electron temporarily occupies an energy state greater than its ground state, it is in an excited state. An electron can become excited if it is given extra energy, such as if it absorbs a photon, or packet of light or collides with a nearby atom or particle. Electrons do not stay in excited states for very long - they soon return to their ground states, emitting a photon with the same energy as the one that was absorbed. It is these millions of photons emitted by atoms which form the radiant energy of light waves.
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Wavelengths and the radiant energy spectrum  The wavelength of a wave is the distance between any two adjacent corresponding locations on the wave train. This distance is usually measured in one of three ways: crest to next crest, trough to next trough, or from the start of a wave cycle to the next starting point. This is shown in the following diagram:

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The lower the wave frequencies the longer the wavelength.

Electromagnetic spectrum

P = 400

Visible light
P = 700 nm

Ultraviolet

Infrared

Wavelength (Angstrom Units)

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Reflected light

Colour and light absorption

Transmitted light

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Colorimetry


In the standard series colorimetric method, the analyte solution is diluted to a certain volume (usually 50 or 100 ml) in a Nessler tube and mixed.


The colour of the solution is compared with a series of standards similarly prepared. The concentration of the analyte equals the concentration of the standard solution whose colour it matches exactly. colours can also be compared to standards via a colorimeter (photometer), comparator, or spectrophotometer.

The possible errors in colorimetric measurements may arise from the following sources:


turbidity, sensitivity of the eye or colour blindness, dilutions, photometer filters, chemical interferences, and variations in temperature or pH.
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Photometry


Photometric methods are based on the absorption of

 

ultraviolet (200400 nm) or Visible (4001,000 nm) radiant energy by a substance in solution.

The amount of energy absorbed is proportional to the concentration of the absorbing substances in solution. Absorption is determined spectrophotometrically or colorimetrically. The sensitivity and accuracy of photometric methods must be frequently checked by testing standard solutions in order to detect electrical, optical, or mechanical malfunctions in the analytical instrument.
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Beer-Lamberts Law of light absorption

Beer's Law is often written in the form of this equation A = abc as a way of summarizing and quantifying the relationship between the absorbance, the nature of the absorbing chemical, the path length of the solution, and the concentration of the solution. It expresses the ideal situation in which these factors are truly proportional to the absorbance.
"A" = the absorbance which you will measure with an instrument. "c" = concentration measured in molarity. "b" = the path length measured in centimeters. "a" is a proportionality factor called the molar absorptivity which is how much light will be absorbed by 1 cm of a 1 M solution of this chemical. Its value depends on what the chemical is and also on what wavelength (or colour) of light is being used.
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Concentration of a test or reference solution

Light absorbed (absorbance) by a test or reference solution

Concentration of test

=
X Concentration of reference

Absorbance of test Absorbance of reference

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Known reference solutions (standards) can be used to graph a straight line, unknown can be determined using linear regression
0.7 0.6 Abs orbance 0.5 0.4 0.3 0.2 0.1 0 0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration, M

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Spectrophotometry


Spectrophotometry involves analysis by  the measurement of the light absorbed by a solution.  The absorbance is proportional to the concentration of the analyte in solution. Spectrophotometers consist of a  light source (tungsten for VIS or hydrogen or deuterium lamp for UV gives steady white light)  a monochromator - device for selecting the desired wavelength  a sample holder - cuvette  a detector - photoelectric cells that measure the transmitted (unabsorbed) light (phototube, photomultiplier tube, or light sensitive diode)
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Instruments used in colorimetric procedures



Spectrophotometers differ from photometers in that:  Spectrophotometers utilise monochromators to provide narrow band wavelengths  photometers use filters to isolate the desired wavelength region,
 

Filters isolate a wider band of light Less accurate readings

The possibility of errors in spectrophotometric analyses is increased when numerous dilutions are required for an analysis.
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Flame photometry (FP),



use of emission spectroscopy in the ultraviolet and visible regions  To identify and estimate the amounts of various elements which are excited in a flame, an arc or high voltage spark. sample in solution is atomized at constant air pressure and introduced in its entirety into a flame as a fine mist.  The temperature of the flame (1,8003,100K) is kept constant.  The solvent is evaporated  the solid is vaporized and  then dissociated into ground state atoms.
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Flame photometry (FP),



The valence electrons of the ground state atoms are excited by the energy of the flame to higher energy levels and then fall back to the ground state. The intensities of the emitted spectrum lines are determined in the spectrograph or measured directly by a spectrophotometer. The flame photometer is calibrated with standards of known composition and concentration. The intensity of a given spectral line of an unknown can then be correlated with the amount of an element present that emits the specific radiation.
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Physical interferences - solute or solvent effects on the rate of transport of the sample into the flame. Spectral interferences - caused by adjacent line emissions when the element being analyzed has nearly the same wavelength as another element. Monochromators - minimize this interference. Ionization interferences may occur with the higher temperature flames. By adding a second ionizable element, the interferences due to the ionization of the element being determined are minimized.
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An advantage of FP is that the temperature of the flame can be kept more nearly constant than with electric sources. A disadvantage of the method is that the sensitivity of the flame source is many times smaller than that of an electric arc or spark. The flame photometry makes it possible to determine in the laboratory, with great precision and simplicity, [Na], [K] and [Li] in every biological liquid.

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Components of a modern Flame Photometer

energised species pass into a cooler part of the flame, lose energy in the form of light of characteristic wavelength as the atoms return to their "ground state".

OH- or oxides of metals smallest aerosol mist vapourisation Larger droplets

Baffles

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Immunochemistry


immunochemistry - the field of chemistry concerned with chemical processes in immunology (such as chemical studies of antigens and antibodies) An antigen is a molecule that stimulates the production of antibodies. Usually, it is a protein or a polysaccharide, but can be any type of molecule, including small molecules coupled to a protein (carrier). An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target.
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Antibodies


In human: immune system-related proteins called immunoglobulins. Each antibody consists of 4 polypeptides


2 heavy chains and 2 light chains joined to form a "Y" shaped molecule.

Variable region
 

amino acid sequence in the tips of the "Y" varies greatly among different antibodies composed of 110-130 amino acids, give the antibody its specificity for binding antigen. includes the ends of the light and heavy chains.

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Antibodies


Treating the antibody with a protease can cleave this region, producing Fab or fragment antigen binding that include the variable ends of an antibody. Material used for the studies shown below originated from Fab. Constant region


determines the mechanism used to destroy antigen.

Antibodies are divided into five major classes, based on their constant region structure and immune function.


IgM, IgG, IgA, IgD and IgE,

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Antigen-Antibody reactions
NATURE OF ANTIGENANTIBODY REACTIONS A. Lock and Key Concept - The combining site of an antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. Thus, our concept of Ag-Ab reactions is one of a key (i.e. the Ag) which fits into a lock (i.e. the Ab).

Antigen

Fab
Papain Hinge

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Antigen-Antibody reactions
NATURE OF ANTIGENANTIBODY REACTIONS B. Non-covalent Bonds - The bonds that hold the Ag in the antibody combining site are all non-covalent in nature.


Antigen

H bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds. Multiple bonding between the Ag and the Ab ensures that the Ag will be bound tightly to the Ab.

Fab
Papain Hinge

C. Reversible - Since Ag-Ab reactions occur via non-covalent bonds, they are by their nature reversible.
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Turbidimetry
When a diluted antigen solution is mixed with a corresponding antibody, antigen Turbidimetry


antibody

immune complexes. (turbid solution)

based on an optical detection system that measure the turbidity, the concentration of very small particles suspended in a solution. A light beam (incoming light) sent trough a solution is scattered depending on the degree of turbidity. A photodetector measures the reduction in the intensity of the light beam (transmitted light).

Since the transmitted light represents a decreasing signal (the more turbid the solution, the more light will be absorbed), one normally refers to the ABSORBANCE this being an increasing quantity in relation to the antigen concentration.
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Electrophoresis
Proteins are composed of amino acids linked by peptide bonds in a sequence and configuration characteristic for each specific protein.
> 100 proteins present in plasma serving numerous physiological functions.

The ability to vary the charge on a protein molecule by changing the pH of its matrix can be used to purify and characterize proteins by electrophoresis and ion exchange chromatography.

When placed in an electric field, molecules with a net charge, such as proteins will move towards one electrode or the other, a phenomenon known as electrophoresis.
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Electrophoresis
Electrophoresis is the movement of charged particles through an electrical field. In order for electrophoresis to occur,  there must be an electrical field,  supplied by providing a tank through which current may pass.  a medium for absorbing and holding the analyte and  charged particles.  supplied by using an appropriate buffer for ionizing the molecule.  After migration the proteins are stained, and protein bands are identified and quantified.
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Electrophoresis is useful as a diagnostic technique for  the separation of proteins in serum, urine and CSF,  the separation of haemoglobins, and  the separation of serum isoenzymes, i.e. creatine kinase (CK).
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