6
DIRECTIONS FOR LABORATORY WORK
Students are obliged to keep the following rules for safe laboratory work, chemical waste disposal, and
the instructions for giving first aid.
The program of practicals is displayed at web pages of Information System of Masaryk University
(http://is.muni.cz). Supplementary material will be uploaded at web pages of ,,VSBC041c Biochemistry II
­ practice" in Information System. If necessary, instructions that are more detailed will be given at the
beginning of lesson.
Students have to be in the laboratory in time and be prepared for the lesson. At the beginning of practical
lesson, the teacher will check the current knowledge of students. Student not sufficiently prepared will not
be allowed to participate in the lesson and will have to do extra practical lesson at the end of semester. All
absences have to be supported by the letter of apology at Department of Study Affairs. All absences have
to be made up till the deadline given.
Students have to wear laboratory coats. They are also recommended to have protective spectacles.
Smoking, eating, drinking, and chewing gums are prohibited in the laboratory.
Work in the laboratory is prohibited for pregnant women and mothers up 9 months after delivery. Female
students are obliged to notify the teacher about pregnancy.
Any injury has to be immediately reported to the teacher.
A short record about work is made in an exercise book, which is shown to the teacher at the end of
practical lesson.
The next week, a lab report is presented to the teacher's approval and signature. The lab report is written
on free A4 papers and contains the following items: studenťs name, date, chapter number and title, the
name of experiment, brief principle, all results (tables, graphs, equations), all answers to questions from
the lab manual.
General guidelines to practical lessons
Reagents and other laboratory equipment are available on laboratory tables. After work, they should be
placed back to the original place.
When heating a solution, be sure to point a test tube to open space, away from other people.
Pipetting of solutions is performed by pipettors and bottle-top dispensers.
Cleaning glassware is performed by students during lesson. Aqueous solutions of chemicals are poured
down the chemical drain (dark grey sink) together with ample tap water. Rinsed vessels are collected in
a marked container.
Solid waste (filter paper, broken glass) is put into a container made for that purpose.
The workplace must be kept in order; all waste must be immediately removed.
7
SAFETY GUIDELINES FOR HANDLING DANGEROUS SUBSTANCES
Corrosives
Corrosive substances are generally strong acids and strong hydroxides.
Concentrated acetic acid is also a corrosive.
Even dilute solutions of these compounds are harmful, especially for eyes.
When handling corrosives, be sure to wear protective glasses or goggles.
Corrosives are delivered by pipettors; mouth pipetting is absolutely prohibited.
Work with corrosives must be performed on the tiled surface in a fume cupboard.
When heating corrosive liquids, be sure to point a test tube to open space, away from other people.
Before dilution, acid is always slowly poured into water, while gently stirring.
Toxic compounds
Examples of toxic substances in practical lessons are methanol and heavy metal salts.
They are delivered by pipettors; mouth pipetting is absolutely prohibited.
Work with irritating and fuming substances are performed in a fume cupboard.
After work with toxic substances, clean the workplace, rinse glassware, and wash your hands
thoroughly.
Flammable and explosive substances
In practical lessons, students may work with natural gas, hexane, alcohols, and concentrated
acetic acid. Some of these substances can be explosive when mixed with air.
All open flames (gas burners) must be extinguished in the presence of flammable liquids.
The residues of flammable solvents must be collected in labelled containers, never flushed
down the drain.
After the spillage of flammable liquid, extinguish all flames, turn off all electric appliances, and
ventilate the room
If the content of a small vessel flames up, it can be ceased by covering the vessel.
In case of larger fire, alarm everyone, turn off gas and electricity in the laboratory, remove all
flammable substances from the surroundings, and use the carbon dioxide extinguisher.
Biological material
In practical lessons, you will work with biological material.
Blood samples are tested, originated from transfusion station.
Serum or plasma are model samples, prepared by diluting substances in the solution of
albumin or prepared from the horse or pig lyophilised sera (e.g. control serum RU or
Lyonorm P).
Urine and gastric juice samples are model aqueous solutions.
8
All biological material must be considered as potentially infectious and the precautions given for
handling with biological material should be respected:
­ Wear protective gloves.
­ Dispose single-use equipment into labelled containers.
­ Use pipettors with exchangeable tips.
­ All glassware contaminated with biological material must be disinfected (e.g. by 1% Jodonal for 2 h)
or sterilized in autoclave.
­ Laboratory table contaminated with biological material must be disinfected (e.g. by 1% Jodonal).
­ Avoid biting your nails or pencil, rubbing the eyes.
­ Always wear a laboratory coat.
First aid
Small injuries (cut)
Wash the wound with tap water, apply disinfection (Septonex, Ajatin) and cover with sticking plaster.
If bleeding is more intensive, apply compressive bandage and seek medical assistance.
Electric current injuries
Turn off electricity, control breathing and heart action, call medical emergency.
Injuries by corrosives
Wash affected area immediately with running water (do not waste time by taking off clothing)
If corrosive splashes the eye, immediately rinse the eye (lids kept open) with water from eye-fountain
for a few minutes, then seek eye specialist.
If corrosive gets into mouth (no mouth pipetting!), immediately rinse mouth with plenty of water, and
seek medical examination.
Affection by toxic substances
Follow the instructions of teacher. First aid and elimination of toxic effects depend on the type of
substance.
Burning injuries
Cool immediately the affected area by cold water for several minutes.
In case of scald (injury from hot vapour) remove the wet clothing and cool with cold water.
Cover the burned areas with sterile gauze pads.
In case of more severe burnings, call medical emergency.
Contamination with biological material
Apply thoroughly disinfection on all contaminated areas and/or wounds including clothing.
9
1 BLOOD. ANALYZERS. LABORATORY
INVESTIGATION. INTERPRETATION
Topics to be reviewed
Blood and its main components. Plasma and serum. Anticoagulants. Haematocrit. Biochemical analyzers.
Reference values, critical difference, factors affecting pre-analytical variability.
Pre-lab questions
1. Which factors may induce haemolysis during blood collection?
2. How many revolutions per minute are recommended for the centrifugation of full blood?
3. Explain the term of ,,dry chemistry".
4. Haematocrit states the volume ratio of erythrocytes in the whole blood. What volume of plasma can
be theoretically obtained after centrifugation of 2 mL of non-coagulable blood, if haematocrit is 0.4?
5. Give examples of disinfectants applied on skin before blood collection.
Introduction
For accurate appraisal of health state many information should be acquired. Results of laboratory
examinations are considered as the source of valid information needed for the assessment of health state
and the reflection of metabolism changes in body. According to World Health Organisation (WHO)
laboratory examinations provides about 80 % information leading to the assessment of correct diagnosis.
The largest and most important part of the clinical-biochemical examinations concerns blood, blood
serum and plasma analyses. Blood is easily accessible material and its composition reflects the range of
biochemical processes blood is a good indicator of the physiological conditions throughout the body.
Another frequently analysed material is urine. Analyses of the other body fluids (gastric juice, duodenal
juice, amniotic fluid, cerebrospinal fluid, saliva, transpiration, etc.) are demanded only in a chosen
number of patients, their frequency is much lower, and they are often carried out in the special
laboratories. A small number of simple, mostly qualitative tests with direct informative results take place
in the doctor's surgery or at the patienťs bed (bed-side diagnostics, point-of-care testing, near-patient
testing).
1.1 Blood collection
Blood for analysis may be taken from veins, arteries, or capillaries. Venous blood is usually the specimen
of choice, for some analysis and especially in young children is taken capillary blood. The arterial
puncture is used only exceptionally, especially for the blood gas analysis.
10
1.1.1 Venous blood collection
With regard to the method and equipment used for the venipuncture are distinguished two ways of
collection:
* Open collection system ­ phlebotomist is in direct contact with the biological material:
­ blood is drawn through needle directly into a blood tube;
­ blood is drawn into a disposable syringe and by back sticking into a blood tube.
* Closed collection system ­ the sample handling is carried out after the collection directly in the
collection syringe or test tube. Thus, the phlebotomist is protected against the contamination by the
patienťs blood, the biological material is protected against the external contamination and also against
breaking during transport and centrifugation. The individual syringes/evacuated test tubes are marked
by colour according to the additive used (clotting accelerators, separation gel, heparin, EDTA, ...). The
separation gel enables proper separation of serum from the blood coagulum after centrifugation
(creation of interlayer). The collection syringe/test tube can be placed directly into the analyzer. The
used material is easily burnt down.
The collection is carried out:
- into the closed syringe ­ the blood is drawn out with the help of a piston (Fig. 1-1a) or with
immediately before the collection prepared vacuum (pulling the plunger into the base of syringe
and locking it into place, Fig. 1-1b). After the collection, the piston is broken off and the syringe is
transformed into the closed blood tube. The method of collection is adapted to patients with
problematic veins. Vacuum is used in case of strong veins.
- into the evacuated test tube (Fig. 1-1c) ­ the system includes a specific blood-collection needle, a
holder into which the needle is assembled before phlebotomy, and evacuated blood collection tubes.
Fig. 1-1a Fig. 1-1b Fig. 1-1c
Evacuated
test tube
Holder
Double spiked
needle
Haemostatic ventil
11
Specimen collection general instruction
* 10­12 hours before the blood withdrawal patients are not allowed to eat; they have to exclude fat food
and alcohol from their diet.
* In the morning before the blood withdrawal, patient can drink about  L of plain water or bland tea.
* Before the blood withdrawal, the patient should be asking for any allergies to antiseptics, adhesives.
* Standard posture of patient at the blood withdrawal is sitting posture.
* Pumping of the fist before venipuncture should be avoided (results of many tests can be affected).
* The site of injection must be disinfected (Jodonal B, Persteril, Jodisol, Ajatin).
* If the veins are visible, the use of a tourniquet should be minimized.
* If the tourniquet is used (not more than 1 min), it should be released before withdrawal of blood begins.
* Press down on the gauze once the needle is out of the arm (to avoid formation of a haematoma).
1.1.2 Capillary blood collection
Capillary blood is used in case only a small amount of the sample is needed. General instructions for
specimen collection:
* Warm the site of injection (tip of the finger, an earlobe, the heel or big toe of an infant), ensure good
blood supply. Avoid massaging the site.
* The injection site must be disinfected.
* Puncture should be directed onto the outer and upper region of the fingertip, halfway between the centre
of the finger pad and the edge of the fingernail.
* After puncture with the lancet, the first drop should be wiped off (dilution of blood with the tissue
fluid). Gently apply intermittent pressure to the surrounding tissue. Avoid excessive squeezing or
"milking" of the puncture site.
* Blood is usually collected into the special capillary blood test tubes or into the small plastic or glass test
tubes. For strip tests, the drop of blood is applied directly on the paper.
* Following collection press clean gauze sponge on the puncture side.
See video recording and acquaint with the blood withdrawal ways.
Give main advantages of closed collection system.
1.2 Blood processing
Depending upon the tests that have been ordered, blood sample may be processed before it is analyzed.
Most routine laboratory tests are performed on either plasma or serum. When blood is drawn without the
addition of an anticoagulant and allowed to stand, it clots after several minutes, because soluble
fibrinogen is transformed to the insoluble network of fibrin by the clotting mechanism. Serum is
excluded from the blood clot after some time and is obtained from clotted blood by centrifugation.
Sufficient time is needed for complete blood clotting (at room temperature at least 15­30 min).
To prepare plasma an anticoagulant must be added to the specimen during the collection. Centrifugation
of non-clotted blood produces plasma. Blood may be centrifuged immediately after the collection.
12
Plasma or serum should be separated from blood cells or coagulum within 2 hours at the latest (or 1 hour
for the determination of potassium ions).
Centrifuges are used to separate components of a mixture on the basis of particle size or density, For separation of blood cells
from plasma or serum there is recommended relative centrifugal force 1 000­1 200 g (g is relative centrifugal force and is
measured in multiples of the earth's gravitational field) for at least 10­15 minutes. Centrifugation of blood is always performed in
the closed test tubes (to prevent aerosolization of infectious particles) at room temperature or 4 C. Prolonged centrifugation or
higher centrifugal force cause often partial or complete haemolysis.
Materials: Sodium oxalate, lithium oxalate, potassium oxalate, sodium citrate, disodium salt of EDTA (ethylenediaminetetraacetic
acid) ­ all as solid compounds, heparin solution in a vial. Blood drawn into a tube with anticoagulant
added, centrifuge tubes, centrifuge.
1.2.1 Preparation of blood plasma
Anticoagulants are substances, which prevent blood plasma from clotting when added to blood during
the collection procedure. Anticoagulant is placed into the test tube in the form of solution and solvent is
evaporated at room temperature. After the collection, blood is mixed gently with the anticoagulant (5 to
10 inversions). Heparin is very often used in such a way that a few drops of heparin solution are drawn
into a syringe and spread over the inner walls of the syringe by repeated movements of the piston. The
blood sample is subsequently drawn into the syringe. Closed collection systems are filled with
anticoagulants during their production.
Approximate amounts of anticoagulants needed to prevent 1 mL of blood from clotting:
*sodium citrate solution is used in haematological tests; that is mixed with blood in the ratio of 1:10 or 1:5;
#
EDTA.Na2 ... disodium salt of ethylenediaminetetraacetic acid; also dipotassium or tripotassium salts are used
+
heterogeneous mixture of sulphonated mucopolysaccharides, produced by mast cells in the tissues of mammals. It accelerates
the action of antithrombin III, which neutralizes thrombin and thus prevents the formation of fibrin from fibrinogen. The
amount heparin added depends on material of collection test tube (glass, plastic).
When using anticoagulants we have to take into account potential changes in the composition of collected
blood, e.g. all anticoagulants bound Ca2+
and Mg2+
ions. Therefore, for the determination of Ca2+
ions a
specially modified heparin should be used. Moreover, during coagulation, the coagulation factors are
activated and platelets release the contents of their granules into the plasma.
Procedure
About 5 mL of blood, which has been prevented from clotting by the use of some anticoagulant and
collected into centrifugation test tubes, centrifuge for 10 min at 2 500 rpm.
Draw chemical formulas of common anticoagulants and explain how they act.
Consider whether the results of some biochemical determinations can be influenced by the choice of
anticoagulant and illustrate with some examples.
Sodium (lithium, potassium) oxalate 1­2 mg
Sodium citrate* 3 mg
EDTA.Na2 (K2, K3)#
1­2 mg
Heparin+
4­6 IU (liquid), 40­60 IU (dry)
13
Describe the appearance of blood before and after being centrifuged.
Give the main differences in composition of blood plasma and serum.
What does it mean, when we describe plasma/serum as a) haemolytic; b) icteric; c) chylose?
Can be a blood sample intended for the collection of plasma kept at 20 °C?
1.3 Manual methods & pipetting
Manual methods have certainly come a long way. At present, they used various commercial complete kits
(in vitro diagnostics kits, IVD kits) containing most of the components needed to perform the
determination. However, most manual methods are still labour intensive, time consuming, limited in their
reproducibility and are limited in terms of overall throughput. The most significant step in all manual
methods is precise and accurate measuring of small volumes of biological sample, which is always
potentially infectious and dangerous reagents.
1.3.1 Manual air displacement pipettors
Air displacement pipettors have a piston that moves in a cylinder
and always certain volume of air remains between the piston and the
sample liquid (volume of all gases significantly depends on external
pressure and temperature). Therefore, accuracy of pipetting is
affected by atmospheric pressure, temperature, and the nature of the
liquid (density, viscosity and homogeneity).
Mechanical pipettors may be fixed volume (capable of delivering a single factoryset
volume) and adjustable (the volume is determined by the operator across a
particular range of values). Pipettors must be fitted with the correct disposable tips
before use.
Pipetting results can easily be influenced by user technique. The
most common pipetting technique is forward (direct) pipetting
which discharges all the liquid by one full movement of the piston.
The technique employs the blow out function ensuring complete
delivery of the liquid (at the end of pipetting the tip must be empty).
It is suitable for aqueous solutions, like buffers, diluted acids or alkalis or solutions containing small
concentrations of protein or detergent. Pre-rinsing of the tip before the actual pipetting improves the
results, but is very often skipped to save time and fatigue.
Reverse pipetting begins and ends with the tip containing liquid. A selected volume plus an excess is
aspirated into the tip. The delivery is done without blow out, and thus, the excess volume remains in the
tip. It is suitable for heterogeneous samples like blood or serum, viscous, or foaming liquids, or very
small volumes of liquid. The advantage of this pipetting technique is reducing error due to film retention.
Piston
Measured
liquid
Bottom part
of pipettor
Disposable tip
Air space
14
General Pipetting Operational Considerations
Select appropriate tip and make sure that the tip is firmly attached to the tip cone. Tips are designed
for single use.
The temperature of the pipetted solution and the pipettor should be the same.
Hold the pipettor vertically when aspirating the liquid. Once there is liquid in the tip, never lay
pipettors on their sides.
When filling volumes > 10 L, pre-rinse each new tip before aspirating the liquid (by filling and
emptying the tip for three to five times).
When aspirating, try to keep the tip at a constant depth approx. 2­5 mm below the surface of the
liquid. Avoid air bubbles in the tip during aspiration.
When pipetting viscous liquids (e.g. serum, blood), leave tip in the liquid for about 1­2 seconds after
aspirating before withdrawing it.
In adjustable pipettors, it is recommended that higher volumes be set first ­ if going from a lower
volume to a higher volume, first surpass the desired volume, and then slowly decrease the volume
until the required setting is reached.
Always control the operating button slowly and smoothly.
When dispensing, hold tip at an angle, against the inside wall of the vessel/tube if possible.
Use the same pipetting technique, pipettor and type of tips in serial pipetting procedures.
1.3.2 Forward pipetting procedure
Attach the appropriate tip to the pipettor.
Set the pipettor to the desired volume.
Press the operating button to the first stop (1).
Immerse the tip so that 2­3 mm is in the liquid and slowly release the operating button to the ready
position (2). Avoid the creation of air bubbles in the tip (Fig. 1-2a).
Be sure to allow for a waiting period at the end of the aspiration to ensure completion.
Withdraw the tip from the liquid, touching it against the edge of the reservoir to remove excess liquid
(Fig. 1-2b).
Dispense the liquid into the receiving vessel (Fig. 1-2c) by gently pressing the operating button to the
first stop (3). After one second, press the operating button to the second stop (blow out) (3).
Remove the tip from the vessel, sliding it along the wall of the vessel.
Release the operating button to the ready position (4).
Ready position
First stop
Second stop
1 2 3 4
15
1.3.3 Reverse pipetting procedure
Attach the appropriate tip to the pipettor.
Set the pipettor to the desired volume.
Press the operating button to the second stop (1).
Dip the tip into the solution to a depth of about 5 mm and slowly release the operating button to the
ready position (2). This will fill the tip with a volume that is larger than the set volume. Avoid the
creation of air bubbles in the tip (Fig. 1-2a).
Wait 1­2 seconds to reach equilibrium in the tip and withdraw the tip from the liquid, touching it
against the edge of the reservoir to remove excess liquid (Fig. 1-2b).
Dispense the liquid into the receiving vessel (Fig. 1-2c) by depressing the operating button gently and
steadily to the first stop (3). This volume is equal to the set volume.
Hold the button in this position. Some liquid will remain in the tip and this should not be dispensed.
Remove the tip from the vessel, sliding it along the wall of the vessel.
Remove the tip from the receiving vessel, sliding it along the wall of the vessel, without blowing it
out. The liquid remaining in the tip can be delivered into the original vessel by pressing the operating
button to the second stop (4) or discarded together with the tip.
Release the operating button to the ready position (5).
Fig. 1-2a Fig. 1-2b Fig. 1-2c
State the main differences between forward and reverse pipetting techniques and suggest the
technique more suitable for pipetting viscous liquids.
Ready position
First stop
Second stop
1 2 3 4 5
16
1.4 Portable and desktop biochemical analyzers
Several types of desktop or portable biochemical analyzers will be demonstrated in the student labs
during the practical lessons. Some of them (glucometers, Accutrend
, Reflotron
) are based on "dry
chemistry" principles. The reagents are not used in the form of solution, as you can see in the majority of
common analyzers, but all the reagents required for the test are applied to a "dry" carrier material ­ e.g. a
filter paper or carrier film. After the drop of the sample (urine, blood) is placed on the carrier, the reagent
is moistened and the reaction proceeds. Coloration is evaluated by the reflex photometry, when the strip is
inserted into the apparatus.
Accutrend
analyzers (Roche-Diagnostics) are simple manual analyzers, which are
used for quick testing of the chosen parameters from capillary blood by dry chemistry.
Accutrend
GC is designed for the determination of glucose and cholesterol. This
"pocket" apparatus is for the personal use of e.g. for diabetics, for bedside monitoring
or near-patient testing.
Before the examination, the instrument must be calibrated with the help of a coding
strip. It is possible to calibrate the both parameters (glucose and cholesterol) at the
same time. Successful calibration is indicated by a three-digit code, which appears on the display. The
code is loaded and saved.
The measurement is carried out with the use of the test strips Accutrend
Glucose and Accutrend
Cholesterol. After the calibration, we can measure with any test strip, whose code is saved in the
instrument.
When the test strip is inserted, the apparatus is automatically switched and the code of the required
parameter appears.
After that, a drop of blood resulting from the puncture of the finger top is placed on the test zone of the
strip. Then the cover is closed and the sample is measured by the reflex photometry method. The level of
glycaemia is evaluated after 12 seconds, cholesterolaemia within 180 seconds.
The Accutrend
GC apparatus can be also used as an electronic diary. The obtained values of glycaemia
and cholesterolaemia can be saved in the individual directories with the possibility to save also
accompanying information describing the conditions of the measurement (e.g. fasting, after the meal,
disease etc.). The apparatus is able to save up to 50 values of glycaemia and 20 values of
cholesterolaemia.
Reflotron
analyzer (Roche Diagnostics) also belongs to the analyzers
working on the principle of dry chemistry. It is used for quantitative
determination of a wide range of clinical parameters from blood, serum,
plasma, or urine. The system uses reagent Reflotron
strips, different for each
determined parameter, and the Reflotron
reflectance photometer controlled by
a microprocessor. Very good unity between the analytical results obtained by
the analyzer and other routine methods is achieved by the calibration
procedures specifically carried out for each set of strips.
Roche Reflotron
systems determine 17 clinical blood parameters covering
liver and pancreas enzymes, metabolites, blood lipids, haemoglobin and
17
Reticular covering layer
Magnetic band
Separation layer
Transport layer
Layer for preliminary reaction
Reaction layer
potassium. The installation of a separation system in the strips enables to use samples of the whole blood
for the analysis (the consumption of blood for one analysis is 30 L; it is possible to use capillary blood).
Reaction strips Reflotron
have a magnetic band on the bottom side, which contains specific information
about the test: Type of the test, incubation time, wavelength, number of examinations, which should be
done, intervals between the individual measurements, mathematical factor for the calculation of the result
from the measurement of reflectance, conversion factor to SI units, internal code of the test. After
inserting the strip into the instrument, this information controls the following analytical steps.
The detection (upper) area of the strip contains
several compartments. The sample, which
should be analysed, is applied on the reticular
covering layer. It is possible to apply whole
blood, serum, plasma. Only accurately
measured dose of the sample from the
micropipette is always applied. Under the
covering layer, we can find the layer of a
special glass wool, which traps erythrocytes and
the other blood elements.
According to the needs a filtration strip can be placed behind the separation layer for preliminary reaction.
This layer can eliminate the interfering substances, may contain the activator or auxiliary reagent (e.g. the
strip for the determination of creatinine contains two zones for the endogenous creatinine cleavage and
elimination of the endogenous ascorbic acid under the separation zone).
Another zone, so called transport zone, enables the transport of plasma to the actual reaction place. It also
contains glass wool, which differs in the composition and orientation of fibres from the previous, so that
plasma is sucked to this zone by the capillary force. The transport layer thus serves as a sponge and at the
same time as a container for plasma. The reaction layer contains reagent, which is essential for the actual
analytical reaction. It is fixed on the special carrier (cellulose film, silica gel, paper). All the above stated
hydrophilic layers are fixed on the transparent hydrophobic foil, which protects the optical system against
contamination at the same time.
The reaction is started, when the reaction zone is pressed to the reservoir with plasma, which follows the
insertion of the strip into the window of apparatus. Thus, the reagent is moistened with plasma and the
reaction is activated. The basic principal of the majority of determinations are enzymatic coloured
reactions. The resulting coloration is measured by the reflectance photometer directed by the
microprocessor.
The advantages of this analyzer include small consumption of blood, non-invasive way of collection
without further processing. It is easy to displace the apparatus. Therefore, it is highly recommended for
performing of larger screening tests. Disadvantages include much higher costs of the analysis.
Turbox
nephelometer (Orion Diagnostica) is used for specific
determination of serum and urine proteins and is based on the principle of
immunoprecipitation followed by measurement of the resulting turbidity.
The analyzer consists of a keyboard, reading apparatus for magnetic cards,
display, cuvette place with a cover for one cuvette, printer, and measuring
element. Besides the analyzer, the producer delivers also complete reagent
18
sets for the determination of the individual proteins. Each reagent set contains an antibody against the
determined protein (antiserum), calibrator (standard of given concentration of the given protein, buffer
solution and reagent calibration card. The card has a magnetic print, which contains the calibration curve
for the appropriate batch. The information is transferred into the apparatus by slipping the card through
the reading part of the analyzer. The instrument analyzes the measurement with respect to the accurate
criteria provided on the reagent calibration card.
Samples, calibrators and antiserums are pipetted directly into the special cuvettes (supplied with the
instrument), where the immunoprecipitation occurs. After the incubation time, the cuvettes are gradually
placed into the cuvette area, where the measurement takes place and the results are printed by the printer.
The results are provided in the Light Scattering Units (LSU), which are selected units representing the
absolute value of the diffused light, and in the concentration units. Levels, which do not correspond with
the criteria given on the magnetic card, are marked with two stars and the explaining reference.
Turbox
system is used for the determination of a large range of the specific proteins (albumin,
prealbumin, haptoglobin, transferrin, components of the complement C3, C4, orosomucoid etc.).
See video recording and acquaint with the utilization of automatic and portable biochemical
analyzers. What analyzers are based on the principle of dry chemistry?
1.5 Factors influencing results of laboratory examinations
The primary function of clinical laboratories is to provide accurate and timely information, based on in
vitro probes examinations, for the diagnosis, monitoring and treatment of the patient. Qualitative and
quantitative tests in body fluids provide the ground for medical reasoning. Many factors besides disease
affect the composition of body fluids. Ignorance or underestimation of these factors may lead to incorrect
interpretation of laboratory results. Laboratory examination includes three phases:
* Pre-analytical phase ­ patienťs preparation, collection of the biological material and its transport to the
laboratory, preparation of the sample for processing;
* Analytical phase ­ analysis of the sample and evaluation of the result;
* Post-analytical phase ­ validation and the result interpretation.
a) Pre-analytical factors
Probably the most important factors regarding laboratory tests and/or procedures are patient identification
and sample identification. It is very important to identify any patient condition or activity that might
affect the lab test or diagnostic test being performed. It has been estimated that 1/3­2/3 of errors occur in
the pre-analytical phase. The laboratory test value may be affected by physiological variables, sample
collection and handling of samples for testing. Physiological factors that affect laboratory test values fall
two categories ­ those that can be controlled and those that cannot.
19
Not controllable biological factors
Race, ethnicity ­ All humans today are 99.9% genetically identical and differentiation of the effects of
race from those of socioeconomic conditions is often difficult. Nevertheless, various race or ethnic
populations differ in the amount of skeleton and skeletal muscles, in physiological levels of some analytes
or in distribution of some genes.
Gender ­ Until puberty, there are few differences in laboratory data between boys and girls. After
puberty may be concentration of numerous components or analytes greater in men than in women.
Differences in physiological values originate mainly from different distribution of hormones. For
example, the higher activity of enzymes originating from skeletal muscle in men is related to their greater
muscle mass.
Age ­ The most important influences, which determine the overall effect of age, are the degree of sexual
maturity and the amount of skeletal muscle mass of the individual. Many laboratory parameters in
children are lower than adults. Higher physiological values during adolescence reflect e.g. increased
osteoblastic activity during bone development.
Pregnancy ­ Pregnancy causes physiologic changes in many body systems, e.g. changes in production of
hormones and their actions, influence of placenta, transfer analytes from amniotic fluid, etc.
Biorhythms ­ biological rhythms may be linear (age) or cyclic changes of metabolism due to action of
hypothalamus and/or pituitary hormones (e.g. circadian rhythms, menstrual cycle) or due climatic or
seasonal influences (dietary changes, physical activity, exposure to sunshine, temperature and magnitude
of its changes). These cyclical changes may be forecast with some probability.
Controllable biological factors
Body habitus ­ Body weight may affect concentration of some analytes by changing their distribution
volumes. The serum concentration of cholesterol and LDL-cholesterol, triacylglycerols, uric acid, insulin
or cortisol positively correlates wit obesity.
Physical activity ­ The effect of exercise on blood composition depends upon the duration and intensity
of the activity and the physical condition of the patient. Muscular activity has both transient and longer
lasting effects. In general, acute strenuous and exhausting exercise increased anaerobic catabolism, while
prolonged endurance exercise aerobic metabolism. Exercise causes a reduction of cellular ATP, which
increases cellular permeability. The increased permeability causes slight increases in the serum activities
of enzymes and metabolites originating from skeletal muscle. Levels of these substances return to normal
soon after the activity is stopped, with the exception of enzymes, which may remain elevated 24 hours or
more. During exercise is increased metabolic activity of skeletal muscle (utilization of glucose and fatty
acids, production of lactate).
Diet/fasting/starvation ­ Diet, food ingestion, fasting and starvation affect investigated analytes by
different mechanism. It depends on the composition and amount ingested food and beverages. The
hormones and enzymes are released before intake of food and during food intake. The meal rich in
protein increases phosphate, urea and uric acid. The food rich in the lipids decreases ratio of nitrogen
compounds, for examples uric acid. The vegetarian food decreases both total and LDL-cholesterol and
triglycerides, increases total bilirubin and pH of urine. Some food and beverages can affect some
metabolic pathways. For example, if the meal contains caffeine, the concentration of catecholamine,
20
glucose and free fatty acid is increased. Dehydratation may alter test results and make it difficult to draw
the patienťs blood. Patient may drink water to prevent dehydratation before the collection of blood
(coffee is not permitted during the fasting period). Haematocrit and the concentration of many compounds
are increased at dehydratation.
Smoking ­ has undesirable effect on the organism. Smoking may affect the level of many analytes by
nicotine. Smoking affects the metabolism of the glucose and increases the concentration of cholesterol
and triacylglycerols.
Alcohol ­ Alcohol consumption changes biochemical analytes in different ways. It depends on the fact
whether the abuse is chronic or acute. Generally, it affects mainly the metabolism of glucose,
triacylglycerols and increases liver enzymes in blood. Single dose of alcohol (small and medium dose)
minimally affects the investigation. Long-term abuse of alcohol results in hypoglycaemia and keto- and
lactatacidose, the increasing of serum uric acid concentration.
Drugs ­ can affect biochemical investigation by a lot of mechanism. For example ­ drugs induce/inhibit
liver enzymes, affect bonding capacity of transport proteins, cause cytotoxicity or may interfere with the
determination of analytes.
Stress ­ Stress affects the production of hormones that consequently change the metabolism of many
compounds. Stress is often connected with severe illness and with collection of blood at some persons.
The fear accompanying the collection influences concentration of some hormones (e.g. catecholamines).
Anxiety that results in hyperventilation may cause acid-base imbalances, and increased lactate.
Environmental factors ­ These factors can include altitude (above 3 000 m), ambient temperature and
geographic localization ­ countryside or city. Travelling across several time zones affects the circadian
rhythm. It results in a change of some analytes, frequently it is retention of sodium ions and water. These
values regress within 2 days after return.
Mechanical action ­ Muscle trauma, intramuscular injection increase the activity of ALT, AST, CK and
the concentration of myoglobin. The pressure of uterus in high stage of pregnancy increases activity of
ALT, the digital investigation of prostate increases activity of PSA (prostate-specific antigen).
Handling of samples
There are many factors to consider when collecting lab specimens and prior to diagnostic tests. Preparation
of the patient prior to the test or diagnostic measure is vitally important to the results of the test.
Sample collection ­ There are a lot of recommendations. The patients are not allowed to eat
approximately 10­12 hours before blood collection. They have to exclude fat food and alcohol from their
diet. Patients can drink about  L of plain water or bland tea in the morning before the blood collection.
Distorted findings in parameters can occur if patients did not keep fasting. Precautions (e.g. dietary and
others) are recommended for some special investigations or functional tests (for example-PSA can be
positive after riding bicycle). Haemodilution may occur due to drawing the blood sample from the same
arm with an intravenous infusion of fluids running.
The type of blood sample needed is very important. The type of blood depends on the test ordered. Be
sure to collect the specimen in the correct blood tube. Certain blood specimens must be collected in tubes
with no anticoagulant. Some specimens must be collected in a tube with anticoagulants. Be sure to handle
the specimens correctly. Some blood specimens must be gently mixed with the anticoagulant in the tube.
21
Time of the collection ­ Concentration of some substances substantially varies during the day (e.g.
glucose, triacylglycerols, hormones), whereas the concentration of the others throughout the month or a
year. Blood collection is performed usually in the morning hours.
Posture ­ Postural changes (supine to sitting etc.) are known to vary lab results of some analytes. Going
from a supine (lying down on the back) position to standing cause the water to filter from the
intravascular environment into the tissues, resulting in a decrease in plasma volume and an increase in
non-filterable elements, proteins and substances, which are bound to proteins (e.g. total protein, albumin,
enzymes, lipids, Ca2+
, Fe3+
ions) by 5­15 %. It takes about 20­30 minutes to equalize fluid shifts due to
changes in position. Standard posture of patient at the blood withdrawal is sitting posture.
Tourniquet Application ­ If the tourniquet is used (not more than 1 min), it should be released before
withdrawal of blood begins. Pumping of the fist before venipuncture should be avoided (results of many
tests can be affected). Prolonged tourniquet application may cause haemoconcentration of non-filterable
elements (i.e. proteins). The hydrostatic pressure causes some water and filterable elements to leave the
extracellular space. Haemoconcentration may occur due to prolonged tourniquet constriction.
Posture ­ Postural changes (supine to sitting etc.) are known to vary lab results of some analytes. Going
from a supine (lying down on the back) position to standing cause the water to filter from the
intravascular environment into the tissues, resulting in a decrease in plasma volume and an increase in
non-filterable elements, proteins and substances, which are bound to proteins (e.g. total protein, albumin,
enzymes, lipids, Ca2+
, Fe3+
ions) by 5­15 %. It takes about 20­30 minutes to equalize fluid shifts due to
changes in position.
Compression of the vein/finger ­ influences the concentration of blood gases, lactate, and pH.
Haemolysis ­ (visible, if the concentration of haemoglobin > 0.2 g/L) ­ haemolytic sample has increased
concentration of the analytes, whose concentration is high in the erythrocytes and vice versa. Haemolysis
may occur due to rough handling of the sample or from drawing the blood through a small-gauge needle.
The causes of haemolysis: the use of extremely big or small needle, rapid evacuation of the syringe, heavy
shaking of blood in the test tube, moisture in the collection set or in the test tube, detergents in the test
tube, wrong proportion of anticoagulant, maintaining blood in the fridge, on the sun or above the radiator,
centrifugation at high speed of rotation.
Transport of samples
The serum/plasma is preferred for the transport than whole blood samples. Haemolysis can occur during
the transport of whole blood. During transport, blood samples should be kept at 0 C (temperature of
thawing ice).
Stabilization and storage of sample
It depends on the determined analyte. There should be low temperature (4 °C, 20 °C, 80 °C), collection
into thawing ice, protection against light, correction of the pH of the sample, the addition of the
stabilisator.
22
b) Analytical factors
Results of all laboratory tests are affected by many analytical factors/errors, which determine closeness of
measured value to true value. Because "true" value never has been exactly known, it is replaced by an
estimate of true value called traceable value (sometimes called "conventional true value", "assigned
value", "best estimate" of the value, "conventional value", "reference value" or "expected value").
Quantitative analytical results are expressed as "estimated value  uncertainty", where "estimated value"
is the estimate of the traceable value obtained when the analytical method is applied to the test sample.
Among basic analytical characteristics are related quantities precision and trueness.
Precision is the ability of analytical method to produce the same value for replicate measurements of the
same sample (i.e. closeness of agreement between independent test results xi obtained under stipulated
conditions). By other words, precision refers to how large the uncertainty in an experimental quantity is.
Precision is not typically represented as a numerical value (it is a qualitative concept) but is expressed
quantitatively in terms of imprecision, which is computed as an experimental standard deviation SD
(has the same unit as measured quantity) or a relative standard deviation RSD (coefficient of variation
CV) of the measurement results:
1
)(
1
2
n
xx
SD
n
i
i
%)100(
x
SD
CV
where xi ... value of measurement, n ... number of measurements and x ... arithmetic mean:
n
x
n
xxx
x
n
i
i
n 121 ...
Imprecision depends critically on the specified conditions. These conditions depend on the different factors that may be changed
between each test result. Depending on the factors changed, two types of precision can be obtained: the repeatability and the
reproducibility. Repeatability is simply the precision determined under the same conditions of measurement (the same
procedure, the same observer, the same measuring instrument, used under the same conditions, the same location, and repetition
over a short period of time). Term of repeatability is identical with the term of precision within-day. Reproducibility is simply
the precision determined under conditions where the same methods, but different equipments, are used by different operator to
make measurements on identical specimens. In general, the reproducibility gives the largest expected precision because it is
obtained by varying all the factors that may affect to the results.
Precision depends only on the distribution of random errors and does not relate to the true value. Random
errors are statistical fluctuations (in either direction) in the measured data due to the precision limitations
of the measurement device. Random errors usually result from the experimenter's inability to take the
same measurement in exactly the same way to get exact the same number. Fortunately, almost all the
random errors follow a Gaussian-shape "bell" curve, and therefore, can be quantified by the standard
deviation or the coefficient of variation. The effects of random errors can be minimized by taking many
measurements and averaging the results.
Trueness is the closeness of agreement between the average value obtained from a large series of test
results x and an accepted reference value x0. Trueness is a qualitative concept. Its quantitative
counterpart is systematic error, which is called ,,bias". Bias is the difference between the expectation of
23
the test results (the average value of the large series of measurements x ) and an accepted reference value
x0:
bias = )( 0xx bias = %)100(
)(
0
0
x
xx
The causes of bias may be known or unknown but should always be corrected for when present. The
accepted reference value x0 is a value that is accepted as "truth" for a measurable quantity. An accepted
reference value usually is obtained as the mean of a large number of measurements by the reference
method in the reference laboratories.
Accuracy is closeness of the agreement between the result of a measurement and an accepted reference
value. Accuracy is a qualitative concept. Its quantitative counterpart is total error (TE), which is a
combination of both systematic error and random error:
TE = 1.96  SD + bias
Systematic errors in some instances can be identified and corrected but depending on its nature may be
difficult or impossible to identify or quantify. That means that in many cases one can doubt about the
reliability of a particular result of measurement. Unfortunately, not only systematic error can introduce a
doubt about a result of measurement: random errors also may move away a result of measurement from
the true value. Random and systematic errors act together on a measurement result producing an error of
measurement and generating doubt ,,uncertainty" about the true value.
Uncertainty u characterizes the dispersion of the values that could reasonably be attributed to the
measured quantity. Uncertainty is the stated range of the best estimated of any given value within which
that value x may be expected to lie with some expressed degree of confidence (i.e. probability).
The uncertainty of a measurement is found by repeating the measurement enough times to get a good
estimate of the standard deviation SD of the values. Then, any single value has a standard uncertainty
equals to the standard deviation (i.e. numerically u = SD).
However, if the values are averaged, then the mean measurement value has a much smaller uncertainty, equal to the standard
error of the mean, which is the standard deviation divided by the square root of the number of measurements.
Uncertainty of measurement comprises, in general, many components. Some of these components may be
evaluated from the statistical distribution of the results of a series of measurements and can be
characterised by experimental standard deviations. The other components, which can also be
characterised by standard deviations, are evaluated from assumed probability distributions based on
experience or other information. The most important random and systematic components of the
Total error
Random error
Bias
x
Measured values xi
Accepted reference
value
x0
24
uncertainty are standard uncertainties of reproducibility and used calibrator, respectively. Therefore, the
total (combined) standard uncertainty (uc) is estimated by the equation:
2
calibrator
2
ilityreproducibc SDSDu
Then the true value lies with a certain probability in an interval (called an expanded uncertainty) that is
calculated from a standard uncertainty (uc) and a coverage factor k, which is equal to 2 (exactly 1.96) at
confidence interval of 95%:
c2 ux
Using good laboratory practice, the measured laboratory data should contain the information about
expanded uncertainty. In addition, the reference or cut-off values should contain the information about
expanded uncertainty.
1.6 Interpretation of results
Clinical-biochemical examinations provide the clinician with a number of important information to derive
the diagnosis, select the correct treatment, and monitor the course of disease to evaluate prognostic or risk
factors.
1.6.1 Comparison of results with reference interval
Interpretation of the results of the biochemical examinations is most frequently carried out by the
comparison with the reference interval. To follow this decision process, these reference values are
required for all tests performed in the clinical laboratory, not only from healthy individuals but also from
patients with relevant diseases. Epidemiological studies and preventive medicine programs may require
reference values from completely healthy individuals, whereas a clinical decision to diagnose a specific
disease would preferably require sets of reference values, which discriminate between specific disorders.
The reference values are values obtained from the chosen group of individuals ­ reference individuals
(age, sex, and race) with defined state of health.
The term normal value has been used frequently in the past. Confusion arose because the word normal has several very different
connotations, therefore, it is recommended use the term reference values.
Reference individual is an individual selected as basis for comparison with individuals under clinical
investigation, using defined criteria. A set of selection criteria determines which individuals should be
included in the group of reference individuals. Such selection criteria include statements describing the
source population, specification of criteria for health or the disease of interest. Ideally, the group of
reference individuals should be a random sample of all individuals in the parent population who fulfil the
selection criteria. Examples of exclusion criteria for health-associated reference values: pregnancy,
excessive exercise, stress, intake of pharmacologically active agents (alcohol, tobacco, oral
contraceptives, drug treatment for disease), genetically determined risk, obesity, hypertension. Often
separate reference values for sex, age group and other criteria are necessary.
25
The test results (reference values) from this sample group (the reference group) are analyzed statistically
to determine an interval of values that includes a specified percentage of all the values (reference
interval) from the sample group.
Reference interval includes 95% of results of reference group; it means that 5% of the results are not
included (2.5% of the highest values and 2.5 % of the lowest values). The reference interval can be
estimated using parametric or non-parametric statistical method. Using of these methods depends on the
distribution of the reference values.
To estimate the reference intervals, it is necessary to exclude outlying values, to test values (Gaussian or non-Gaussian
distribution), to estimate reference interval using parametric or nonparametric method, to test influence of age/sex.
The parametric method is used for data having Gaussian distribution (normal distribution). The mean ( x )
and standard deviation (SD) are calculated from reference values.
n
x
n
xxx
x
n
i
i
n 121 ...
1
)(
1
2
n
xx
SD
n
i
i
Then the reference interval is calculated
sax
where a is coefficient 2 (exactly 1.96) for 95% interval of reference values, x mean, SD standard deviation.
The parametric method is based on the determination of  2 SD limits (exactly 1.96) from the mean of the
reference distribution. In a Gaussian distribution, these limits correspond to the 0.025 and 0.975 fractiles
(see scheme below).
Many analytes have skewed distribution ­ most frequently with tail towards higher values. In this case, it may be possible to
apply a mathematical transformation (for example, taking logarithm) to reduce the data to Gaussian distribution, but such
transformation may only approximate the ideal. A reference interval, mean and standard deviation will be calculated after log
transforming the observations of the method. The calculated parameters are shown back-transformed into units of the method.
Nonparametric method can be also used for estimation of reference interval. The basic assumption is that
the values do not fit a Gaussian distribution. The non-parametric method is based on sorting the reference
values in ascending order of magnitude. The reference range can be calculated by ranking the values and
deleting the lowest and the highest 2.5 % (see schema below). Such a procedure has the considerable
advantage that it requires no assumptions to be made about the characteristics of the distribution and does
not require any transformation of the data to be made. The major disadvantage of the non-parametric
method is that it cannot be applied to small sets of reference values. The recommended minimum number
of reference values is 120.
The interpretation of the laboratory results with the help of the 95% interval of the reference values is the
expression of the probability at the same time. Healthy person has 95% probability that his test result will
lie in the given reference interval. The remaining 5 % represent the probability, that the result will be
lower (2.5 %) or higher (2.5 %) than the reference interval.
It is necessary to verify the validity of the reference intervals taken from the literature (for given method in laboratory and for
given population).
26
Parametric and non-parametric method used for estimation of reference interval:
The probability, that the test results of all reference individuals will lie within the reference
intervals, decreases with the increasing number of test results. Thus, the probability that the result of
healthy individual will lie out of reference range increases.
1.6.2 Decision-making analysis
Another interpretation of the results is so called decision-making analysis. In this type of evaluation, we
do not use the interval of reference values ("from-to"), but only one cut-off value (decision limit). This
value allows us to describe the determined result as the positive test (the values are higher usually than
cut-off value) or as the negative test (usually cut-off value or lower values). The positivity of the test is
connected with a certain risk of existing or future disease. The risk could be described verbally
(increased, high, etc.) or numerically (probability in percentage, etc.).
The cut-off value is usually calculated in such way, that the maximum of the so-called substance of the test is achieved, i.e. the
highest probability of the unity of the test with the diagnosis. The ideas and their practical application will be explained in the
clinical biochemistry more closely.
In case the patients are monitored during the progression of the disease or the medical therapy, the
laboratory findings are compared with the previous results. This procedure does not enable the
mechanical comparison of results, but the analytical and biological variability must be taken into account.
1.6.3 Critical difference
Most clinical laboratory tests are not used to aid in the diagnostic process but in monitoring. Data on
biological variation are required for the interpretation of changes in serial results. A change may be due
not only to the patient improving or deteriorating but also to analytical imprecision and intra-individual
biological variation. Therefore, for a change to be significant, it must exceed the so-called critical
difference.
Critical difference (CD) or reference change value (RCV) is expressed as statistically significant
difference between the two results of the given laboratory test measured in an individual between the
given time interval. If data on analytical imprecision, i.e. analytical variation CVa (repeatability between
days), and intra-individual (within-subject) biological variation CVi are known, then the critical difference
can be calculated as:
Values are ordered according to their size from xmin to xmax
Frequency
Median
SDx 96.1
2.5 % tail area
(1/40)
P = 5 %
2.5 %
97.5 %
27
2
i
2
a77.2 CVCVCD
where the factor 2.77 is derived from 1.96 2, where 1.96 is determined by the 95% confidence interval values and 2 arises as
we are comparing two results obtained by the same method for given analyte (i.e. results with the same CV).
For current database of biologic variations, see http://www.westgard.com/biodatabase1.htm. Calculator of CD is possible to find
on the internet ­ see web pages of Czech Society of Clinical Biochemistry http://www.cskb.cz.
If the difference of two results of the given laboratory test in an individual measured between some
definite time interval is higher then calculated CD, it can be expected that the results are different. These
results differ with a certain probability (usually 95 %), i.e. they are not caused by the analytical or
biological variability, but they reflect the change of the clinical state of the patient.
If the difference of the two measurements is lower than the acceptable CD value, the arithmetic mean is
calculated for the given parameter. If the critical difference is crossed, the third examination is carried out
after some time.
1.6.4 Diagnostic accuracy
The basic idea of diagnostic test interpretation is to calculate the probability a patient has a disease under
consideration given a certain test result. We test some people for the presence of a disease. Some of these
people have the disease, and our test says they are positive. They are called true positives (TP). Some
have the disease, but the test claims they do not. They are called false negatives (FN). Some do not have
the disease, and the test says they do not ­ true negatives (TN). Finally, we might have healthy people
who have a positive test result false positives (FP). Thus, the number of true positives, false negatives,
true negatives, and false positives add up to 100 % of the set. We can assume that there are four possible
groups of patients ­ see Fig. 1-3 and the truth table.
Fig. 1-3: Distribution of individuals with regard to presence of the disease and results of laboratory test
TN is a negative test for patients who do not have the disease, FN is a negative test for patients who have the disease,
TP is a positive test for patients who have the disease, FP is a positive for patients test who do not have the disease.
Number of
patients
Result of test
Disease
No
disease
Cut-off value
TN
TP
FN FP
Negative test Positive test
TN ... True negativity
FN ... False negativity
TP ... True positivity
FP ... False positivity
28
Truth table (2 x 2 table): Classification of patients
Number of subjects tested
Test result
Diseased No Diseased Totals
Positive test TP FP TP + FP
Negative test FN TN TN + FN
Totals TP + FN TN + FP TP + TN + FP + FN
Diagnostic accuracy is the ability of the test to discriminate between two states, i.e. disease and no
disease, and is described by two key parameters: diagnostic (clinical) sensitivity and specificity.
Sensitivity refers to the proportion of people with disease who have a positive test result. Sensitivity is
probability that diagnostic test will indicate presence of disease when disease is actually present. This
term essentially tells the clinician how good the test is at correctly identifyingpatients with disease.
%)100(
FNTP
TP
ysensitivitDiagnostic
Sensitivity is the percentage of diseased individuals who have a positive test. Sensitivity is also the truepositive
rate, e.g. if the sensitivity is 95 %, 5 out of 100 individuals with the disease will test negative. If
the sensitivity is 100 % the test recognizes all sick people as such (100 % true positives, 0 % false
positives).
Specificity refers to the proportion of people without disease who have a negative test result. Specificity
is probability that diagnostic test will indicate absence of disease when disease is actually absent. This
term essentially tells the clinician how good the test is at correctly identifying the absence of disease.
%)100(
FPTN
TN
yspecificitDiagnostic
Specificity is the percentage of healthy individuals who have a negative test. Specificity is also the truenegative
rate, and the inverse, 1 specificity, is the false positive rate. If the specificity is 95 =, 5 out of
100 individuals without disease will test positive. If the specificity is 100 % test recognizes all healthy
people as healthy (100 % true negatives, 0 % false negatives).
The values of both parameters are changed in the range 0­1 (0­100 %). The closer the sensitivity or
specificity is to 100 %, the more sensitive or specific the test.
Sensitivity is the ability of a test to classify correctly an individual as "diseased". Specificity is the ability
of the test to classify correctly an individual as "disease-free". Both sensitivity and specificity describe
how well the test discriminates between patients with and without disease. An ideal test could distinguish
absolutely between patients who do and who do not have disease (sensitivity and specificity equals 1).
The clinical usefulness of a test is determined by how much it deviates from this ideal. A test where both
sensitivity and specificity are close to 1 has good diagnostic ability.
Sensitivity and specificity of given test vary depending on the analyte cut-off level selected to distinguish
positive from negative results. When the cut-off value is lowered (i.e. the cut-off line is moved to the
left), more diseased individuals are classified as diseased and sensitivity is increased. On the other hand,
29
if the cut-off value is raise i.e. the cut-off line is moved to the right), more non-diseased individuals are
classified as healthy. Sensitivity and specificity are inversely proportional, meaning that as the sensitivity
increases (less false negative results, lower FN), the specificity decreases (more false positive results,
higher FP) and vice versa.
High sensitivity is required when early diagnosis and treatment is beneficial, and when the disease is
infectious. High specificity is important when the treatment or diagnosis is harmful to the patient mentally
and/or physically.
1.7 Test requisition forms
The sample of blood or other biological material is sent for examination to the central clinicalbiochemical
laboratory in the majority of cases. The required examinations are marked into the laboratory
order ­ the test requisition form. The requisition form should contain appropriate information about
patient (patient name, birth identification number, diagnosis code), ordering physician (ID of health
institution, name and qualification number of physician), billing (insurance identification), specimen
(source, date of collection) and selected tests (all tests being ordered). Laboratory orders should be
entered (electronically or on paper) directly by the physician. In some hospitals and outreach laboratories,
laboratory tests are requested on paper requisitions, which are physically sent to the laboratory for entry.
The results together with their interpretation (numerical, graphic, and verbal) are then sent back
(electronically or on paper) in the test results document. Different workplaces use different design of
formularies.
Familiarize yourselves with the requisition forms used in several hospitals and how to fill out them.
Which sections are included in all formularies?
Note the results of the performed examinations into the results document too in course of the
semester.
Related problems
1. Mr. Z. has is going to come to venipuncture on the next day. Describe, what rules should he
follow 24 hours before the collection.
2. Mr. S is a race swimmer. He came to the morning venipuncture after two hours training in the
swimming pool. What analytes could be affected by the physical strain?
3. Miss M. (22 years, league basketball player) came to the regular check-up connected with the
venipuncture. She had to stand in the overcrowded tram for one hour before she came to the
doctor and she was not allowed to sit in the waiting room either. Analysis of her plasma
showed, that the concentration of the total protein was at the upper limit and the level of
albumin was slightly raised. Both values were found normal during the second control after two
weeks. What can explain the previous results?