Relation between pathobiochemistry and
clinical biochemistry. Clinical-biochemical
analytics and its specific features. Terminology
of clinical biochemistry. Material analyzed.
Material collection.
Relation between pathobiochemistry and clinical biochemistry
• Pathobiochemistry = a field dealing with biochemical processes
under pathological conditions/biochemical mechanisms that lead to
the development of a certain disease.
• Clinical biochemistry = interdisciplinary field focused on research of
basic biochemical processes in human organism from the point of
view of human health resp. from the point of view of the treatment
of diseases, eventually to prevent the onset of the disease.
Clinical-biochemical analytics
• Biochemical analyte = investigated biochemical substance.
• The analytes may be of endogenous or exogenous origin.
• Analytes of endogenous origin: substrates and products of cellular
metabolism: ions, minerals, enzymes, vitamins, trace elements and other
substances that are physiologically present in the human body.
• Analytes of exogenous origin: substances of natural origin (toxins of animals,
fungi, plants, moulds, bacteria, allergens), drugs and their metabolites,
alcohols, addictive substances, substances abused for doping, industrial
products, industrial waste.
• Laboratory analysis of the analyte is carried out by a well-defined
examination method and consists of 3 parts: pre-analytical,
analytical and post-analytical phases.
Preanalytical phase
• Preparation of patient for biological sample collection.
• Biological sample collection.
• Pre-analysis adjustment of the sample after collection (addition of an anticoagulant - obtaining non-clotting blood, NaF for
glucose testing in plasma, conservants…).
• Fill in the request form.
• Sample transport to clinical laboratory.
• Receiving of the sample.
• Separation of serum/plasma from the cellular component of blood.
• Storage of samples/aliquots (if the sample is not immediately examined).
• Temporary archiving of sample for possible repeat analysis.
• Preparation of analytical samples by physical and chemical adjustment from biological samples (pH adjustment, concentration,
dilution, dialysis, sedimentation, filtration, extraction into organic phase, protein denaturation, mineralization, amplification molecular
biological examination, drying, homogenization, lyophilization…).
• Non-standard conditions of the preanalytical phase (incorrect collection of sample, unacceptable transport time, unclear sample
identification, insufficiently filled request form, spilled tube contents…) → clinical laboratories are obliged to reject the material.
Analytical phase
• Preparation of the analytical system: start-up of laboratory
instruments, tempering water baths, performing of the optical tests
and calibration, internal quality control).
• Analysis of the biological sample by an appropriate examination
method.
• Conversion of specific physicochemical quantities (absorbance,
fluorescence, chemiluminescence, etc.) to analyte concentrations
expressed in usual units.
Postanalytic phase
• Expression of the obtained result, rounded to the appropriate number of
decimal places, and insertion of the obtained values into the formulas (e.g.
for the calculation of renal clearance).
• Interpretation of the result with respect to the physiological range of
values, diagnosis, stage of the disease, stage of its treatment and also
analyte values measured at the previous collection.
• Confirmation of the result by medical laboratory technician and clinical
biochemist.
• Sending the result (printed/electronic) to the doctor.
• In some cases, the result may be delivered directly to the patient or
persons legally authorized to take it over.
Preanalytical influences on the laboratory tests
• The results of the laboratory examination can be influenced in all
three phases.
• The most important from the point of view of influencing the results
is the preanalytical phase.
• In the preanalytical phase the result may be influenced by:
• Person of patient
• Collection of a sample
• Transport of sample
• By storing the sample before analysis
• Sample preparation for processing
Person of patient-controllable factors
• Gender (most laboratory tests do not depend on gender, but we also find methods where the reference
range differs for women and men, e.g. red blood cell parameters).
• Race, ethnic or social groups of population (e.g. negroid races have significantly less granulocytes than
europoid race, they also differ in reference values ​​of other tests such as amylase, alkaline phosphatase,
creatine kinase). Members of ethnic groups may differ, for example, in the frequency of certain genes and
thus in the occurrence of hereditary metabolic disorders. The reference values ​​may also be influenced by
typical eating habits of some ethnic or social group.
• Age: most tests have a lower upper limit of the reference range in childhood. E.g. higher activity of acid
alkaline phosphatase and inorganic phosphorus concentration due to bone formation. The results of some
laboratory tests (e.g. creatinine clearance) in children should be corrected according body weight or body
surface area.
• Simultaneous other disease (e.g. in a patient with rheumatoid arthritis, we find an elevated c-reactive
protein).
• The half-life of the analyte (e.g. diagnosis of myocardial infarction according to enzyme activity and levels of
muscle protein in serum). Knowledge of the rate of elimination of a substance from the body is of great
importance in the evaluation of drug levels and toxic substances = half-life of xenobiotics.
• Method of setting reference values.
Person of patient-controllable factors
• Pregnancy: proteins and other substances produced by trophoblast
or fetal organs (hCG, SP-1, α1-fetoprotein, placental alkaline
phosphatase, estrogens…) appear in the mother's blood and urine.
Hormonal effects are also applied to some common metabolites:
cholesterolemia increases in the last trimester, hemoglobin
concentration decreases due to hemodilution, increased rate of
glomerular filtration → decreased creatinine and urea in serum…
• Cyclic changes (e.g. diural rhythm of cortisol, monthly rhythm of
female sex hormone secretion) The concentration of some other
analytes is influenced by circadian rhythm, too.
Person of patient-uncontrollable factors
• Physical activity (e.g. Increased concentration of total protein and substances bound to
it, pH changes (under anaerobic load), lactate concentration increases, muscle proteins
are released into the blood circulation (increased activity of CK, AST, LD, myoglobin
concentration)… changes depend on a number of factors such as the duration of
exercise, exercise intensity (aerobic, anaerobic), individual training.
• Psychological stress leads to release of adrenal hormones and their metabolic effects,
e.g. hyperglycemia, increase of free fatty acid concentration…
• Food, alcohol, fluid intake.
• Smoking (e.g. may affect the rate of metabolism of the drug theophylline).
• Drugs: the drug can affect the metabolism of an analyte (it changes its rate e.g. by
inducing synthesis or inhibiting enzymes, affects binding to transport proteins…) or
interferes with its own chemical reaction (e.g. ascorbic acid may mask the presence of
glucose/blood in urine.
• Surgery: narcotics (hepatotoxicity), contusion and damage of tissue (increased CK, AST,
LD, myoglobin concentration), hormonal response to stress.
Basic samples of biological material
• Whole blood
• Serum
• Plasma
• Urine (single, collected)
• Sweat
• Gastric content
• Cerebrospinal fluid
• Exudation and other body fluids
• Stool
• Bone marrow
Collection of sample
• It depends on the patient's position for some time before and during collection
and the type of collected blood. Additives to the collected blood and the type of
container may also influence the result.
• Collection of venous blood: from the elbow vein.
• Patient position: lying down is recommended. Standing up there is a transfer of fluid from
the intravasal space to the interstitium and the concentration of high-molecular substances
(and substances bound to them) in the blood, including hematocrit, can increase by 10 - 15%.
• Skin disinfection: use of alcoholic disinfectant solution before blood collection to determine
alcohol concentration → false positive result. Skin disinfection with surfactant (Ajatin) →
haemolysis.
• Arm tightening + exercise should be as short as possible, otherwise the above fluid
movement, anaerobic metabolism (local acidosis) may occur.
• Beware of vacuum during blood collection. Vacuum too high → mechanical hemodialysis.
• Collection of other types of blood: capillary blood (e.g. from the fingertip for blood glucose
measurements), arterial blood (blood gas testing), arterialized capillary blood (acid-base
balance).
Containers/tubes
• Fill in etiquette of conatainers (tubes) – name of patient → identification of
patient. It is also possible to identify the patient using a code, e.g. Bar code.
• Drain blood with the syringe slowly, never through the needle (to avoid
hemolysis).
• Most examinations are performed from serum → blood must be clotted.
• One-use only containers are the most convenient. They are mostly made of
plastic (blood clots slowly and insufficiently, haemolysis occurs more often) →
the tubes with a layer of kaolin or glass wool → acceleration of precipitation. A
special internal gel allows separation of serum (gel forms the interface between
the blood cells and the serum layer).
• Glass tubes → better blood clotting, BUT: possibiltiy of a rupture of tubes in
centrifugation (specimen degradation, centrifuge contamination). Before reuse,
the container must be decontaminated and washed → reagent residue in the
test tube → devalued assay results.
Examination from the whole blood and plasma
• It is important to maintain the prescribed ratio between the volume of
anticoagulant solution and the added blood.
• Blood is always added to the tube with the anticoagulant, never vice versa.
• The anticoagulant influences the composition of the collected blood. E.g. all
anticoagulants, including heparin, bind Ca2+ and Mg2 + and decrease their
concentration. To determine the Ca2+ concentration we must use heparin titrated
by these ions. Various enzymes may be inhibited.
• All anticoagulants are anions where the accompanying cation is Na+ (K+, NH4
+, Li+).
E.g. for the determination of ions from collected blood in a tube designed for the
determination of blood glucose (contains NaF and Na2EDTA), we get the
increased value of Na+ concentration.
• Examples of analytes: levels of glucose, lactate, glutathion, acid-base balance,
glycated hemoglobin (whole blood), fibrinogen, acid phosphatase (plasma).
Sample transport
• The blood is transported in closed containers.
• Protect samples of blood during transport from extreme
temperatures (inactivation of enzymes in the heat, glucose
concentration decreases faster, frost can cause hemolysis).
• Exposure of blood to light may cause degradation of bilirubin.
• Transport must be fast enough (1-2 hours from collection depending
on the analyte).
• We always prefer to send serum over longer distances than whole
blood (because mechanical haemolysis).
Storage of samples
• Storage conditions affect the stability of the analytes.
• Prolonged standing of the collected blood leads to depletion of energy sources of erythrocytes
(glucose) → impaired membrane function (ensures transport of Na+ out and K+ to the cell) → K+
leakage from erythrocytes → hyperkalaemia.
• Heat storage → leukocytes consume oxygen + CO2 production + anaerobic glycolysis in
erythrocytes → decreased blood pH. Therefore, blood for examination of acid-base balance and
blood gases is kept on ice.
• Addition of NaF in the blood for glucose/lactate assay → inhibition of glycolysis. Without the
addition of NaF, blood glucose decreases by erythrocyte activity.
• Most examinations are performed from serum. Store in a fridge at + 4 °C in a well-closed tube to
avoid evaporation of the sample (analytes and enzymes are stable for several days). Prolonged
storage → storage of frozen serum (at - 20 °C/- 80°C).
• Chemical conversants are rarely used to stabilize serum (they should not interfere with the
analysis). They are used for conversation of urine samples → we always choose them according to
the type of analysis. Examples of chemical conversants: thymol in isopropanol, HCl, formalin (cell
fixation).
Processing and sample preparation for analysis
• Centrifugation of blood. Too strong → haemolysis of blood!
• Separation of serum/plasma.
• Deproteinization of the sample (usually using chemical reagent →
select according to subsequent analysis).
• Thickening of the sample (e.g. concentration of proteins contained in
urine/cerebrospinal fluid prior to their separation by electrophoresis,
choosing the right pore size).
• More rarely other procedures (e.g. erythrocyte washing).
Hemolysis
• = disintegration of erythrocytes + spillage of their contents, including red blood agent into
plasma (serum).
• Haemolysis usually occurs in vitro, i.e. during blood collection, transport and processing.
• According to the cause we divide hemolysis into:
• Mechanical (too strong shaking, centrifugation at high speed, transporting whole blood over a long distance,
etc.)
• Osmotic (wet tube)
• Thermal (blood exposed to frost/high temperature).
• Chemical (disinfectant that disrupts the erythrocyte membrane).
• Influence of results by hemolysis:
• Intracellular erythrocyte content is released from the erythrocytes into the plasma → increased
concentration of intracellular components (e.g. LD, AST, ACP, potassium).
• The red color of hemoglobin interferes with the photometric determination of analytes.
• Hemoglobin acts as a buffer and changes the pH of the reagent → it affects the reaction (e.g. determination
of albumin, hemolysis decreases the result).
Examination methods in clinical biochemistry
• Laboratory observations:
• analyte examined by the human senses (sight, smell).
• Often a microscope is used, such as the microscopic determination of urinary tract
stones.
• Laboratory observation = qualitative analysis → we evaluate what is present in the
sample, not expressing the amount, content or concentration of the analyte.
• Qualitative biochemical methods belong to the Identification methods → we perform
the detection of the analyte = we determine the presence/absence of the analyte in
the sample.
Examination methods in clinical biochemistry
• Laboratory measurements:
• The analysis is performed in biochemical analyzers.
• Examination based on scanning physical or chemical quantities using detectors → laboratory measurements.
• Instrumental examination methods: optical methods (spectrophotometry in UV-VIS, reflex photometry, atomic
absorption spectrometry, luminescence spectrometry, turbidimetry, nephelometry), separation (chromatography,
electrophoresis, mass spectrometry, ultracentrifugation), electrochemical (potentiometry, amperometry) ,
physical (osmometry, flow cytometry) and microscopic (diascopic and episcopic microscopy).
• Laboratory measurement of a qualitative character: the measurement result corresponds to the positive negative
response of the detector. E.g. Detection of gene mutation by real-time PCR, in laboratory finding we evaluate
presence absence of mutation.
• Laboratory measurements of quantitative character: the detector response is proportional to the intensity of the
measured physico-chemical quantity (Lambert-Beer's law) in a certain measuring interval. If we have a sufficiently
large number of calibration standards, we can determine by their analysis the dependence of the measured
quantity on the analyte concentration → get the calibration equation → determination of the analyte
concentration. The result is expressed numerically and is provided with units (mmol/l, μmol/l, g/l, mg/l, μkat/l).
We determine the concentration/amount of the analyte of interest in the sample.
• Laboratory measurements of semiquantitative character: we do not have a reference sample (calibrator) for the
analyte evaluation → we are not able to define the equation of the calibration curve. We can only estimate the
concentration, whether it is pathological, physiological or low, moderate, high (based on experience, reliability of
instruments and reference samples containing a known amount of a substance with similar physico-chemical and
biological properties as the analyte of interest). E.g. immunochemical analysis of benzodiazepines (the device is
calibrated for one drug from the benzodiazepine family and other drugs in this group are tested with less
reliability).
References:
• BERÁNEK, Martin a Miloš TICHÝ. Vybrané kapitoly z klinické
biochemie: pro studijní program Zdravotnická bioanalytika. Praha:
Karolinum, 2013. ISBN 978-80-246-2186-9.
• RACEK, Jaroslav. Klinická biochemie. 2., přeprac. vyd. Praha: Galén,
c2006. ISBN 80-726-2324-9.
• http://lekarske.slovniky.cz/pojem/patobiochemie
• https://cs.wikipedia.org/wiki/Klinická_biochemie