3 Bio Chem Analysis

8/3/2019 3 Bio Chem Analysis

1/47

INTRODUCTIONTO

BIOCHEMICAL ANALYSIS

Lecture 3

Basic Chemical Pathology

MPAK 1112


2/47

2/14/2012 2

Brief history of development:(1) Mid 19th century - HCl in gastric juice, urine tests for sugar, protein and bile.

(2) 1920s - venepuncture routine practice, colorimeters widely available, bloodanalyses from wards and side rooms to hospital laboratories.

(3) 1940 and 1950s - advancement of instrumentation and development of micro-and ultra-micro methods ===> steep increase in variety of and demand forchemical pathology investigations.

(4) 1960 and 1970s - new tests + automatic equipment capable of multipleanalysis, e.g. 240 samples per hour, 30 tests per sample ===> computerizeddata processing ===> screening and profile tests.

(5)

1980s - consideration of finance (escalating cost), efficacy and priority(allocation of resources: one patient over-investigated may have to meananother underinvestigated) ===> control of unrestricted demands for analysis,improvement on SELECTIVITY in the choice and frequency of investigations.

(6) 1990s - trend in 1980s continues with further cost-containment;multidisciplinary laboratory automated systems.


3/47

DIAGNOSTIC TESTS:Basic Principles, Use and Interpretation


4/47

2/14/2012 4

Diagnostic tests

Assists the clinician1. Screening to identify risk factors for disease

early intervention to prevent disease occurrence

to detect occult disease in asymptomaticpersons. 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


5/47

2/14/2012 5

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.


6/47

2/14/2012 6

What is biochemical testing?

Biochemical testing looks at the levels of specific

substances and enzymes that are produced by chemicalreactions 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 itsusual chemical reactions.

As a result, several harmful changes in bodybiochemistry can occur such as accumulation ofsubstrate, accumulation of precursors, deficiency ofproducts, redirection of substrate into alternatepathways.


7/47

2/14/2012 7

What is biochemical testing?

Biochemical testing looks at the levels of thesedisturbances and can be used to detect severaldifferent metabolic conditions.

The analysis of these constituents can be eitherqualitative or quantitative


8/47

2/14/2012 8

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 aspectrophotometer

Flame emission spectrometry - a substance is forcedunder pressure into a flame which then emits light tant can bemeasured by a photosensitive detector


9/47

2/14/2012 9

Laboratory tests

A laboratory test: a procedure in which a sample of blood, urine or

other tissues or substances in the body ischecked. often part of the routine examination to detect

possible changes in an individuals health before anysigns and symptoms appear.

play an important role in diagnosis Symptoms present

to plan treatment, monitor and evaluate theresponse to treatment, or monitor the course of

illness over a period of time.


10/47

2/14/2012 10

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 orthe back of the wrist with a needle.


11/47

2/14/2012 11

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 testsare usually carried out in hospitals. If a small blood sample is required e.g. blood glucose

estimations, a few drops of blood can be squeezedout from a tiny prick on the fingertip or ear lobe.


12/47

2/14/2012 12

Samples for examinations in a biochemistry laboratory

Serum the fluid remaining after blood has

clotted and the clot has been removed. the clot - the red blood cells, white

blood cells, and platelets Plasma

the fluid which remains after the redcells have been removed from blood

clotting has been prevented by use ofanticoagulant

Urine Appearance, SG, microscopy

Faeces

Gastrointestinal blood, Cerebrospinal fluid (CSF)

the fluid made in the choroid plexus of the ventricles in thebrain and secreted into the subarachnoid space around thebrain and spinal chord.

Specimens for lab examinations are obtained from lumbar

puncture


13/47

2/14/2012 13

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/thumbas this will alter some constituents)

Collect volumes up to about 200 QL Alternative containers: capillary tubes


14/47

2/14/2012 14

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 flowinto 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 transportspecimens to the lab Swabs, needles, syringes,

Containers


15/47

2/14/2012 15

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 toproteins UV/daylight eg destroys bilirubin Preservatives to prevent bacterial growth esp in

urine samples


16/47

2/14/2012 16

Special Considerations for Biochemical Testing

Technical considerations Labelling specimens

Label accurately information shouldcorrespond 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


17/47

2/14/2012 17

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 Atoms of the same element that have the same

number of protons but different number of neutrons

(same atomic number but different atomic mass)


18/47

2/14/2012 18

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 reductionOIL 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


19/47

2/14/2012 19

General Principles of Biochemical Analyses

Every manipulation in biochemical analysis is a potential

source of error so that in the interest of accuracy andprecision the number of manipulations be kept to aminimum.

There are 6 basic operations in each biochemicalanalysis:1. A measured aliquot of the sample is deproteinised2. A measured volume of the protein free filtrate or supernatant is

transferred to the reaction tube3. Measured volumes of the reagents are then added

4. Samples and reagents are thoroughly mixed5. The reaction is allowed to proceed for a specific time6. A product of the reaction i.e colour, fluorescence or electrical

signal is measured for the sample, standards and blank.


20/47

2/14/2012 20

General Principles of Biochemical Analyses

Possible worst causes of error: Measurement of volumes of sample and

reagents

Timing of the reaction


21/47

2/14/2012 21

Analytical Techniques

Photometry Colorimetry

Spectrophotometry

Flame Photometry

Electrochemistry

Turbidimetry

Electrophoresis

Immunochemistry


22/47

2/14/2012 22

Principle of Colorimetric Analysis

Colorimetry

Compares the colour produced by an unknownquantity of a substance with the colourproduced by a standard containing a knownquantity of that substance.

Basis of the principle of colorometric analysis: Many substances are coloured in solution

Intensity of colour in these solutions is related tothe amount of substance in solution

Low concentration pale colour

High concentration darker colour

The coloured solutions can absorb light at givenwavelengths in the visible spectrum.


23/47

2/14/2012 23

Origin & nature of light energy

Light energy

Source electron activity

The ground state of an electron, theenergy level it normally occupies, is the

state of lowest energy for that electron

There is also a maximum energy that eachelectron can have and still be part of its

atom. Beyond that energy, the electron isno longer bound to the nucleus of the atomand it is considered to be ionized.

eorbit


24/47

2/14/2012 24

When an electron temporarily occupies an

energy state greater than its ground state,it is in an excited state. An electron canbecome excited if it is given extra energy,such as if it absorbs a photon, or packet oflight or collides with a nearby atom orparticle.

Electrons do not stay in excited states forvery long - they soon return to theirground states, emitting a photon with thesame energy as the one that was absorbed.

It is these millions of photons emitted byatoms which form the radiant energy oflight waves.


25/47

2/14/2012 25

Wavelengths and the radiant energy spectrum

The wavelength of a wave is the distance between anytwo adjacent corresponding locations on the wavetrain. This distance is usually measured in one of threeways: crest to next crest, trough to next trough, or

from the start of a wave cycle to the next startingpoint. This is shown in the following diagram:


26/47

2/14/2012 26

The lower the wave frequencies the longer the wavelength.

Electromagnetic spectrum

Wavelength (Angstrom Units)

Visible light

Ultraviolet Infrared

P = 400 P = 700 nm


27/47

2/14/2012 27

Transmitted

light

Reflectedlight

Colour and light absorption


28/47

2/14/2012 28

Colorimetry

In the standard series colorimetric method, the analyte

solution is diluted to a certain volume (usually 50 or 100ml) 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 mayarise from the following sources: turbidity, sensitivity of the eye or colour blindness, dilutions,

photometer filters, chemical interferences, and variations intemperature or pH.


29/47

2/14/2012 29

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 theconcentration of the absorbing substances in solution.

Absorption is determined spectrophotometrically orcolorimetrically.

The sensitivity and accuracy of photometric methodsmust be frequently checked by testing standard solutionsin order to detect electrical, optical, or mechanicalmalfunctions in the analytical instrument.


30/47

2/14/2012 30

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 therelationship between the absorbance, the nature of theabsorbing chemical, the path length of the solution, andthe concentration of the solution. It expresses the idealsituation 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 thischemical. Its value depends on what the chemical is and also onwhat wavelength (or colour) of light is being used.


31/47

2/14/2012 31

Concentration of atest or referencesolution

Light absorbed (absorbance) bya test or reference solution=

Concentration of test

Absorbance of test X Concentration of referenceAbsorbance of reference

=


32/47

2/14/2012 32

Known reference solutions (standards) can beused to graph a straight line, unknown can bedetermined using linear regression

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 0.1 0.2 0.3 0.4 0.5 0.6

Concentration, M

Absorbance


33/47

2/14/2012 33

Spectrophotometry

Spectrophotometry involves analysis by

the measurement of the light absorbed by a solution. The absorbance is proportional to the concentrationof the analyte in solution.

Spectrophotometers consist of a light source (tungsten for VIS or hydrogen or

deuterium lamp for UV gives steadywhite light)

a monochromator - device for selecting the desiredwavelength

a sample holder - cuvette a detector - photoelectric cells that measure the

transmitted (unabsorbed) light(phototube, photomultiplier tube, or lightsensitive diode)


34/47

2/14/2012 34

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 analysesis increased when numerous dilutions are required for ananalysis.


35/47

2/14/2012 35

Flame photometry (FP),

use of emission spectroscopy in the ultraviolet and

visible regions To identify and estimate the amounts of various

elements which are excitedin a flame, an arc or high voltage spark.

sample in solution is atomized at constant airpressure and introduced in its entirety into a flameas 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.


36/47

2/14/2012 36

Flame photometry (FP),

The valence electrons of the ground state

atoms are excited by the energy of theflame to higher energy levels and then fallback to the ground state.

The intensities of the emitted spectrum

lines are determined in the spectrograph ormeasured 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 theamount of an element present that emits

the specific radiation.


37/47

2/14/2012 37

Physical interferences - solute or solvent effectson the rate of transport of the sample into theflame.

Spectral interferences - caused by adjacent line

emissions when the element being analyzed hasnearly the same wavelength as another element.

Monochromators - minimize this interference.

Ionization interferences may occur with the

higher temperature flames. By adding a secondionizable element, the interferences due to theionization of the element being determined areminimized.


38/47

2/14/2012 38

An advantage of FP is that the temperature ofthe flame can be kept more nearly constant thanwith electric sources.

A disadvantage of the method is that the

sensitivity of the flame source is many timessmaller than that of an electric arc or spark.

The flame photometry makes it possible todetermine in the laboratory, with great precision

and simplicity, [Na], [K] and [Li] in everybiological liquid.


39/47

2/14/2012 39

Components of a modern Flame Photometer

vapourisation

smallestaerosolmist

Larger droplets

Baffles

OH-or

oxides of

metals

energised species pass intoa cooler part of the flame,lose energy in the form oflight of characteristicwavelength as the atomsreturn to their "groundstate".


40/47

2/14/2012 40

Immunochemistry

immunochemistry - the field of

chemistry concerned with chemicalprocesses in immunology (such aschemical studies of antigens andantibodies)

An antigen is a molecule that

stimulates the production ofantibodies. Usually, it is a protein or apolysaccharide, but can be any type ofmolecule, including small moleculescoupled to a protein (carrier).

An antibody is a protein used by theimmune system to identify andneutralize foreign objects like bacteriaand viruses. Each antibody recognizes aspecific antigen unique to its target.


41/47

2/14/2012 41

Antibodies

In human: immune system-relatedproteins called immunoglobulins.

Each antibody consists of 4polypeptides 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.

b d


42/47

2/14/2012 42

Antibodies

Treating the antibody with aprotease can cleave this region,producing Fab or fragment antigenbinding that include the variableends of an antibody. Material usedfor the studies shown beloworiginated from Fab.

Constant region determines the mechanism used to destroy antigen.

Antibodies are divided into five major classes, basedon their constant region structure and immunefunction. IgM, IgG, IgA, IgD and IgE,


43/47

2/14/2012 43

Antigen-Antibody reactions

NATURE OF ANTIGEN-ANTIBODY REACTIONS

A. Lock and Key Concept - Thecombining site of an antibody islocated in the Fab portion of the

molecule and is constructed fromthe hypervariable regions of theheavy and light chains. Thus, ourconcept of Ag-Ab reactions is oneof a key (i.e. the Ag) which fitsinto a lock (i.e. the Ab).

Antigen

FabPapain

Hinge


44/47

2/14/2012 44

Antigen-Antibody reactions

NATURE OF ANTIGEN-

ANTIBODY REACTIONSB. Non-covalent Bonds - The

bonds that hold the Ag in theantibody combining site are allnon-covalent in nature. H bonds, electrostatic bonds, Van

derWaals forces and hydrophobicbonds.

Multiple bonding between the Ag andthe Ab ensures that the Ag will be

bound tightly to the Ab.C. Reversible - Since Ag-Abreactions occur via non-covalentbonds, they are by their naturereversible.

Antigen

FabPapain

Hinge

T bidi t


45/47

2/14/2012 45

Turbidimetry

When a diluted antigen solution is mixed with a corresponding

antibody,

antigen antibody immune complexes.(turbid solution)

Turbidimetry 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 (themore turbid the solution, the more light will be absorbed), onenormally refers to the ABSORBANCE this being an increasingquantity in relation to the antigen concentration.


46/47

2/14/2012 46

Electrophoresis

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 byelectrophoresis and ion exchange chromatography.

When placed in an electric field, molecules with a netcharge, such as proteins will move towards one electrode or

the other,a phenomenon known as electrophoresis.

Proteins are composed of amino acids

linked by peptide bonds in a sequenceand configuration characteristic foreach specific protein.> 100 proteins present in plasma serving

numerous physiological functions.


47/47

2/14/2012 47

Electrophoresis

Electrophoresis is useful as adiagnostic 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).

Electrophoresis is the movement

of charged particles throughan electrical field.In order for electrophoresis to

occur, there must be an electrical

field, supplied by providing a

tank through whichcurrent may pass.

a medium for absorbing andholding the analyte and

charged particles. supplied by using an

appropriate buffer forionizing the molecule.

After migration the proteinsare stained, and proteinbands are identified andquantified.

Documents

3 Bio Chem Analysis