Physiopathology
(Clinical
Biochemistry)
Serkan SAYINER, DVM PhD. Assist. Prof.
Near East University, Faculty of Veterinary Medicine Department of Biochemisty
serkan.sayiner@neu.edu.tr
What will we learn in Clinical Biochemistry?
▪ What is Clinical Biochemistry?
▪ Interests of Clinical biochemistry
▪ Biological Materials
▪ Pre-Analytical Phase (Technical and Biological Factors)
▪ Analytical Phase (Method selection, method validation, quality control)
▪ Post-Analytical Phase (Evaluation of results, reference value, reporting)
What will we learn in Clinical Biochemistry?
▪ Fluid-Electrolyte Balance
▪ Acid-Base Balance
▪ Clinical Enzymology
▪ Liver Function Tests
▪ Pancreas Function Tests
▪ Kidney Function Tests
▪ Thyroid and Parathyroid
▪ Glandula suprarenalis, pituitary gland
▪ Digestive functions (Ruminants, rumen?)
▪ Plasma and serum proteins
▪ Blood Lipids
▪ Skeletal Muscle
▪ Reproductive Endocrinology
▪ Tumor markers
References
▪ Karagül H, Altıntaş A, Fidancı UR, Sel T, 2000. Klinik Biyokimya. Medisan, Ankara
▪ Kaneko JJ, Harvey JW, Bruss ML, 2008. Clinical Biochemistry of Domestic Animals, 6th edi.
Academic Press-Elsevier
▪ Thrall MA, Weiser G, Allison RW, Campbell TW, 2012. Veterinary Hematology and Clinical Biochemistry, 2nd edi. Wiley-Blackwell
▪ Pineda MH, Dooley MP, 2003. McDonald’s Veterinary Endocrinology and Reproduction, 5th edi. Blackwell Publishing.
▪ Rizzi TE, Valenciano A, Bowles M, Cowell R, Tyler R, DeNicola DB, 2017. Atlas of Canine and Feline Urinanalysis, 1st edi. Wiley-Blackwell
▪ Sink CA, Weinstein NM, 2012. Practical Veterinary Urinanalysis, 1st ed. Wiley-Blackwell
What is Clinical
Biochemistry, what are
its areas of interest?
Definition of Clinical Biochemistry and Physiopathology
▪ The Physiopatholgy means the functional changes associated with or resulting from disease or injury. It examines physiological changes that accompany a particular disease. In other words; It is a physiology that occurs in an abnormal internal environment or metabolic reactions.
▪ CLINICAL BIOCHEMISTRY is a science that examines the body and its various parts of fluid and tissue (biopsies) taken or discarded from the body in terms of diagnosis and course of disease. Results is also used in monitoring and determining treatment procedure and prognosis.
▪ Briefly; It is a clinical-specific laboratory science.
▪ It is a kind of clinical pathology branch that aims to interpret the body
fluids, tissues and cells and to interpret the health and disease relation of the test results.
Clinical Biochemistry
▪ Clinical biochemistry examines abnormal mechanisms that normally deviate from the molecular level.
▪ Laboratory techniques and molecular mechanisms developed for this work are used.
▪ It reaches the target indirectly, but thanks to the developing technologies, it gives very specific and sensitive results.
▪ Chemical analyzes are mainly performed in serum or plasma.
Clinical Biochemistry
▪ It is aimed to give information to the students who are trained in
veterinary medicine to gather information on the theoretical and practical knowledge of Biochemistry on the case and if necessary to explain all the information and skills that can be used in clarifying the molecular
mechanisms of the disease or disorders and in the selection of the
parameters which may be helpful in diagnosing the disease and in the interpretation of the test results.
▪ Clinical biochemical tests play an important role on the path to disease diagnosis. 1 out of 3 laboratory tests performed in hospitals are clinical biochemical tests.
Clinical Biochemistry
▪ In addition to the functions it undertakes in diagnosis and prognosis, there is also an important role in preventive medicine.
▪ It helps to evaluate animal nutrition and aquaculture problems, to observe differences and relations or interactions between species,
breeds, physiological and regional conditions, to determine population normals.
▪ Clinical biochemistry and biochemistry are directly related to clinical medicine and many other scientific disciplines.
Biochemistry and
Clinical Biochemistry
Genetics
Molecular Biology
Toxicology
Immunology Microbiology
Virology Endocrinology
Physiology
Pharmacology
Veterinary Medicine
Human
Medicine
History
Examination
Diagnostic Services
Laboratory Service
Clinical Biochemistry
Radiological services, Physiological test such as EEG, ECG, Pulmonary Function Test
Emergency Test Routine Test Special Tests
Haematology Histopathology Cytology Immunology Microbiology
Biological Molecules Investigated in
Clinical Biochemistry
Metabolites
▪ They are end products or resultant molecules or sometime intermediate molecules of metabolic reactions.
▪ Concentration measurements give information about metabolism.
▪ Their concentrations are expressed in
conventional or SI units in serum or plasma.
▪ Conventional: mg/dL, g/dL
▪ SI: mmol/L
▪ Approximately 60% of clinical biochemistry analyzes involve metabolites.
▪ Eg: Urea, Creatinine, Albumin, Protein-Total, Bilirubin, Cholesterol
Enzymes
▪ They are normally intracellular protein molecules.
▪ Except functional enzymes found in plasma (!).
▪ High activity generally refers to cellular degeneration, expect functional plasma enzymes.
▪ 30% of clinical biochemical analyzes include enzymes.
▪ Eg.: AST, ALT, GGT, CK, LDH ....
Minerals
▪ Macro and Micro (trace) elements.
▪ They have important functions in body fluids and tissues such as energy production, balance in osmotic pressure, acid base balance, neural
function, to be a cofactor in enzyme activation etc.
▪ Ör.: P, Ca, Na, K, Cl, Zn, Cu, Co, Fe ....
Süt humması Source: UWaterloo
Hormones, Vitamins and Others
▪ They are found in trace amounts in body fluids and tissues.
▪ Serum or plasma levels are in the ng, pg or nmol, pmol level.
▪ The functions they involve are extremely important.
▪ About 10% of the biochemical analyzes include these molecules
▪ Eg.: Steroid hormones (such as Progesteron), 25-OH Vitamin D3,Total T4
Biological Materials
Blood, Urine, Stool, Lymph, Sweat, Tear, Digestive Secretions, Puncture Fluids, CSF, Genital Secretions, Stones And Tissues
Blood
▪ It is a biological fluid circulating in the vascular system.
▪ Contents: Molecules in solute state + cellular components in suspended state
Source: FisherBioservices
Blood
▪ Chemical Composition: 85% water, 15% solids.
▪ Rates vary between animals, metabolic status, and pathological conditions.
▪ Functions
▪ It is a transport system.
▪ Maintain pH, osmotic pressure balance in tissues and organs.
▪ Protect body temperature.
▪ Providing a defense mechanism against infections.
▪ Oxygen transport etc.
Blood
▪ When should the blood sample be collected?
▪ Usually after 12 hours fasting (???).
▪ What is the difference between Serum, plasma and whole blood?
▪ There is fibrinogen in the plasma.
▪ Which type of sample is obtained from what type of tube?
Blood
▪ Collection
sites: Artery, Vein, Capillary
?
Source: WikiMedia
Blood
▪ Blood Collection: Artery, Vein, Capillary?
▪ Ruminants: Vena jugularis, vena caudalis mediana (coccyeal vena)
▪ Cats and dogs: Vena cephalica antebrachii, vena saphena lateralis, vena saphena medialis, vena jugularis
▪ Chickens: Vena ulnaris
▪ Rabbit: Vena auricularis
▪ Rodentler: Vena caudalis, heart, juguler vein
▪ Other Reptiles: Vena jugularis, vena caudalis, vena cephalica, cervical venous sinus
▪ We have to determine in advance what type of blood samples
we should collect by using what type of blood tube?
Blood
Source: Town Center Vet
Blood
Source: Flickr
Source: The Cat Clinic Source: Metzer Farms
Blood
Kaynak: Zwarg ve ark., 2014 Kaynak: Reptiles Magazine
Urine
▪ What is urine? Define it.
▪ It is Ultrafiltration product of blood or plasma secreted from the kidneys.
▪ The blood is filtered through the glomerulus (~1700 L).
▪ In the proximal tubules, electrolytes, glucose and amino acids are reabsorbed.
▪ Na, Cl, and H
2O are reabsorbed in the connective tubes
(ADH?).
Urine
▪ Functions
▪ Maintenance of balance between extracellular and intracellular fluids.
▪ Maintain acid-base balance.
▪ Removal of metabolite products from the body.
▪ Contents
▪ Water (~95%) and salts (Na, Cl, K, Ca, Mg ...)
▪ Acids and bases (H +, OH-)
▪ Degradation products during metabolism (such as urea, uric acid, creatinine)
▪ Toxic or detoxified substances
▪ Substances that are found hyper levels in blood (such
as glucose, acetone, bilirubin ...) Source: NatureWorlds
Urine
▪ Sample collection
▪ Must be sterile (avoid
contamination) and adequate amount.
▪ Collection from the ground?
▪ Spot urine (free capture)
▪ By using a catheter
▪ Cystocentesis
Source: University of Minnesota
Urine
▪ Urine Analysis or Urinalysis
1. Physical Examination
▪ Color, Odor, Turbidity
▪ Quantity, Density, pH
2. Chemical Examination
▪ Glucose, Protein, Ketone, Bilirubin, Urobilinogen, Nitrite, Blood
3. Microscopic Examination: It’s an examination of urine sediment under microscope (at 40X high power field).
Sediment is obtained by centrifugation.
▪ Organic sediments
▪ Inorganic sediments
Urine
Stool
▪ It is also called faeces.
▪ Physical and chemical examinations can be done to obtain information about metabolism and a possible pathological condition, especially in GI tract.
▪ Frequent tests are fecal occult blood (FOB), determination of bile pigments, organic acids,
pancreatic amylase, elastase, fat droplets and starch
▪ Apart from these, cells and/or parasites can be examined.
Other Biological Materials
▪ Lymph
▪ Sweat
▪ Tear fluid
▪ Humor aqueous
▪ Digestive secretions
▪ Saliva
▪ Puncture fluids (Synovial fluid, cyst fluid, amniotic fluid, CSF)
▪ Stones (Kidney, Pancreas, Bile)
▪ Tissues
Source: TodaysVet.Pra.
Analytical
Phase Post-
Analytical
Phase
Pre-Analytical Phase
Technical and Biological Factors
Pre-Analytical Phase
▪ It is the process that begins with the animal's history (anamnesis) and ends with the start of the requested laboratory test.
▪ In medical laboratories, blood samples are taken by trained personnel and the patients are guided according to the tests requested and samples are collected under appropriate
conditions.
▪ In the VETERINARY LABORATORY, the pre-analytical phase has more limited scope. A part of the pre-analytical phase (may be the biggest part) is completely outside the control of the
laboratory. Blood collecting stage is especially performed in
clinics.
Pre-Analytical Phase
▪ Pre-Analytical phase is divided into two stages in veterinary medicine.
1. Pre-laboratory stage: Mainly includes animal preparation, sample collection and transportion to laboratory.
2. In-Laboratory stage: Starts after sample arrived to laboratory.
▪ It is known that 2/3 of the errors in veterinary clinical laboratories are occured in this phase.
Therefore, it is called pre-analytical errors (Hooijberg
ve ark., 2012) .
Pre-Analytical Phase
▪During sample collection, it should be taken into account that some factors may affect
animals. Like,
▪ The starvation or satiety status.
▪ Stress factors like traveling or being brought to
a new clinical enviroments etc.
Pre-Analytical Phase
Incorrectly collected or poor quality samples will lead to errors such as
•Mistakes in the results,
•Re-collecting of samples
•Incorrect evaluation of test results,
•Inefficient use of personnel and financial
resources.
Pre-Analytical Phase
1.TECHNICAL FACTORS
2.BIOLOGICAL FACTORS
Pre-Analytical Phase – Technical Factors
▪Technical factors include collecting of
samples using appropriate techniques and materials, storage conditions and
transmission to the laboratory according to
the required test or tests.
Pre-Analytical Phase – Technical Factors
Choosing the right type of blood collection tube
▪ Whole Blood: Blood sample is collected to tubes containing
Anticoagulant matter.
▪ It is mainly used for hematological tests, especially complete blood count
(CBC)/hemogram. Also for blood group determination, preparing blood film, direct coombs test, immunologic test.
▪ In mammalians, Ethylenediaminetetraacetic acid (EDTA) is used in dry form (as dipotassium (K2) salt sprayed in a tube) or in liquid form (as tripotassium (K3)).
Today, plastic K2EDTA tubes are mostly used.
▪ In some mammalians, especially in cats, EDTA causes aggregation of platelets.
▪ For birds, reptiles and other species, blood collection tubes containing lithium heparin is recommended.
▪ Another heparinized blood collection tube which has limited use is a dark green capped tube and contains sodium heparin.
Pre-Analytical Phase – Technical Factors
▪ SERUM and PLAZMA: Serum or heparin plasma is recommended for the analysis of many biochemical parameters.
▪ There are no anticoagulant matters in these tubes.
▪ The most commonly used tubes are called serum separator tubes
(SST) to obtain serum. They contain a gel (thixotropic gel) and clotting activators on the tube wall (inner surface) which facilitate seperation of serum and prevent re-mixing by entering between blood cellular elements and serum. These tubes are also called serum separator tubes (SST). They are gold tapped tubes.
▪ Another tube type used to obtain serum is a non-gel containing tube.
These tubes are supplied with a dry tube name and usually have a red tap.
▪ Similar results are usually obtained by using heparin plasma and serum in biochemical test parameters.
Pre-Analytical Phase – Technical Factors
▪ In the analysis of clotting factors, tubes containing citrate as
anticoagulant should be used. These tubes have a light blue tap.
▪ Other than these tubes, there are also tubes which are used for
different and special purposes. For example, the gray tapped sodium fluoride/potassium oxalate-containing tube is preferred for glucose and ketone analysis. This tube is called the glucose tube.
▪ There are also tubes that are not used or used rarely in the field of veterinary medicine but which are used in human medicine.
▪ Ex. dark blue tapped contaminant-free tube for trace element
analysis.
Pre-Analytical Phase – Technical Factors
Choosing the Right Blood Collection Tube
▪ Blood collection tubes are produced from glass or plastic material.
▪ Today, plastic tubes are used more often for safety reasons.
▪ Before colletcing blood samples, the label information on the tube should be read to check the expiration date and it the date has passed, it should not be used.
▪ Plastic anticoagulant tubes should also be assessed to detect a mark on the tube indicating how much blood should be taken.
▪ Use of expired blood tubes and/or collecting inaccurate amount of blood will affect test results. These are
frequently encountered pre-analytical errors in clinical laboratories.
!
Pre-Analytical Phase – Technical Factors
BLOOD COLLECTING TECHNIQUES
In large animals, there is usually no significant difference between blood sampling from different large vessels for routine
hematological and biochemical tests.
There may be differences in the results obtained from blood samples taken from different veins in rodents and cats.
Blood samples collected from the tail veins in cows may be in the form of a mixture of venous and arterial blood, which may be
effective in measuring blood gases.
Pre-Analytical Phase – Technical Factors
▪ Specimens taken from the back of the ear (auricular vein) in cats and dogs differ from those taken from large veins.
▪ If the right techniques and materials are not used, or if the wrong venipuncture technique is applied, tissue damage, hematoma formation, influences of clotting initiation, hemolysis and enzyme activities may lead to erroneous results (increases).
!
Pre-Analytical Phase – Technical Factors
Pre-Analytical Phase – Technical Factors
Pre-Analytical Phase – Technical Factors
URINE SAMPLES
▪ Urine collection is usually done by free-catch sampling during urination a clean container.
▪ The container must be a sterile urine collecting container.
▪ If bacteriological tests are to be performed, aseptic
techniques such as catheter or cystocentesis should be applied.
▪ While spot urine specimens could be collected in animals, it
was very difficult to collect 24-hour urine specimens.
Pre-Analytical Phase – Technical Factors
SALIVA
▪ Veterinary routine use is not common.
▪ It can be an alternative for the measurements of some parameters, such as cortisol and the diagnosis of feline leukemia (FeLV).
STOOL (Faeces)
▪ Some metabolic and pathological conditions can be determined by physical and chemical examination of the stool.
▪ The stool sample should be taken in a clean container and fresh samples should be sent to the laboratory.
▪ Fecal occult blood (FOB), digestive ferments, and endocrine markers (hormones) in wild animals are some stool test examples.
Pre-Analytical Phase – Technical Factors
CEREBROSPINAL FLUID (CSF)
▪ The content of organic and inorganic matters is lower than that of serum. It is very poor for proteins. Glucose
concentration of CSF is about 60% lower than blood glucose concentration. CSF Ca is 50% lower than blood Ca.
▪ The region of CSF sample collection in cats and dogs is
important. In CSF from the lumbal region, the concentration of protein is two times higher than that from the atlanto-
occipital region.
Pre-Analytical Phase – Technical Factors
SYNOVIAL FLUID
▪ There may be differences in the chemical and cellular
components of the synovial samples collected from different joint regions. Iatrogenic haemorrhage may occur due to
incorrect technique.
PERITONAL FLUID
▪ In the horse, protein analysis and cell count may rise after
laparotomy or castration.
Pre-Analytical Phase – Technical Factors
Pre-Analytical Phase – Technical Factors
TEMPERATURE
▪ Generally, samples are stable at room temperature for 2 hours and analysis results can be determined at optimal levels.
▪ For light sensitive analytes such as bilirubin, the samples should be kept in the dark conditions.
▪ In hematological tests to be performed with whole
blood, samples should not be frozen.
Pre-Analytical Phase – Technical Factors
TEMPERATURE
▪ Samples must be stored at –20 ºC if it is wanted to keep it for a long time.
o It is important to know how long each test parameter can be maintained by freezing.
o For example, albumin in serum can be stored for 2
months at -20 °C, while plasma ammonia is only 7 days.
Urine and stool specimens delivered to the
laboratory should always be freshly collected.
Pre-Analytical Phase – Technical Factors
CENTRIFUGE
▪ A centrifuge is a laboratory device that is used for the separation of fluids, gas or liquid, based on density.
Separation is achieved by spinning a vessel containing
material at high speed; the centrifugal force pushes heavier materials to the outside of the vessel.
▪ In clinical laboratories, it is used to separate serum and/or plasma. The force is expressed as RCF (relative
centrifugal force) and is given as a multiple of the acceleration of gravity (g).
▪ The recommended RCF and duration for plasma and serum
separation is 1500-2000g x 10 minutes.
Pre-Analytical Phase – Technical Factors
CENTRIFUGE
▪ RCF or RPM?
oRCF=1.118 x 10
-5x r x rmp
2▪ In cytological samples, a lower g force is used to increase cell concentration and preserve cell morphology.
▪ Since there is no relationship between the number of cells
and crystals and speed or force in the urine samples, 400-
3900 g x 5 minutes can be used.
Pre-Analytical Phase – Technical Factors
Pre-Analytical Phase – Technical Factors
Source: Kaneko ve ark., 2008
Pre-Analytical Phase – Technical Factors
SPECIMEN CONTROL BEFORE ANALYSIS
▪ Specimen quality is an important issue and it must be checked.
▪ Firstly, transport process is assessed.
▪ The presence of hemolysis, lipemia, icterus is checked.
▪ HEMOLYSIS is the destruction of red blood cells which leads to the release of hemoglobin from within the red blood cells into the blood plasma. According to degree of hemolysis, colour of serum changes from light to dark red. Hemolysis is the most frequently observed error in the pre-analytical phase both in human and veterinary laboratories.
▪ Especially in spectrophotometric analyzes it causes false results.
Pre-Analytical Phase – Technical Factors
Hemolysis Index
Source: EclinPath
Pre-Analytical Phase – Technical Factors
Hemolysis Index False Increase False Decrease
≥ 49 Direct Bilirubin
≥ 80 Fe Direct Bilirubin, Bile Acids
≥ 200 AST, Cholinesterase, CK, Fe, LDH, K HCO3-, Direct Bilirubin, Bile Acids
≥ 300 AST, Cholinesterase, CK, Fe, LDH, K, P
HCO3-, Direct Bilirubin, Bile Acids GGT
≥ 400
AST, Cholinesterase, CK, Fe, LDH, K,
P, Mg, Total Protein, Triglycerides HCO3-, Direct Bilirubin,GGT, Bile Acids, Amylase
≥ 600
AST, Cholinesterase, CK, Fe, LDH, K, P, Mg, Total Protein, Triglycerides,
Cholesterol
HCO3-, Direct Bilirubin,GGT, Amylase, Bile Acids, ALP
≥ 800
AST, Cholinesterase, CK, Fe, LDH, K, P, Mg, Total Protein, Triglycerides,
Cholesterol
HCO3-, Direct Bilirubin, GGT, Amylase, Bile Acids, ALP, NEFA
Note: There may be differences between species.
Pre-Analytical Phase – Technical Factors
▪ LIPEMIA occur due to increased chylomicrons and/or very low density lipoproteins in plasma.
▪ This can be seen in patients with hyperlipidaemia or in patients that post-
prandial samples are collected. Lipemic serum and plasma appearance vary from hazy to creamy. Sample can, in some cases, interfere with accurate measurement of clinical pathologic analytes through various
mechanisms.
Source: CLSI
Pre-Analytical Phase – Technical Factors
Lipemia Index False Increase False Decrease
≥ 50 Bile acids
≥ 100 Bile acids, direct bilirubin Na, K, Cl
≥ 200 Bile acids, direct bilirubin Na, K, Cl
≥ 500 Bile acids, direct bilirubin, Mg Na, K, Cl, HCO3-
≥ 1000 Bile acids, direct bilirubin, Mg, Amylase Na, K, Cl, HCO3-
Note: There may be differences between species. It may also vary depending on the concentration of triglycerides.
Pre-Analytical Phase – Technical Factors
▪ The icterus index can be used to determine if there is
hyperbilirubinemia, i.e. if the total bilirubin is increased, the icteric index should closely match the value seen.
Icteric Index False Increase False Decrease
≥ 9 Cholesterol, Triglycerides, Creatinine, Bile Acids
≥ 19 Cholesterol, Triglycerides, Creatinine, Bile Acids,
Total Protein
≥ 40 Mg Cholesterol, Triglycerides, Creatinine, Bile Acids,
Total Protein, GGT, Lipase, ,Uric Acid
≥ 55 Mg Cholesterol, Triglycerides, Creatinine, Bile Acids,
Total Protein, Lipase, Uric Acid, Amylase
Note: There may be differences between species.
Source: Jashnani ve ark., 2012
Pre-Analytical Phase – Technical Factors
▪ The most common error in blood collected in EDTA or heparinized tubes for haematological examinations is the presence of clots. It is the most rejection reason together with haemolysis.
▪ The clots may be visible or they may be in the form of micro clots that may not be seen even when carefully controlled.
▪ Micro clots can affect cell count and clotting factors, as well as time loss and additional expenditure by clogging device probes.
▪ Micro clots and microscopic platelet clusters are common in cat
specimens. In dogs, it is rare.
Pre-Analytical Phase – Biological Factors
▪Biological Factors: Contains more
comprehensive factors than technical factors.
▪ These include factors such as fasting/postprandial, stress, exercise, species (sometimes breed),
gestation, gender, milk yield, climate and environmental conditions.
▪ It is very challenging to control biological factors,
especially in veterinary medicine.
Pre-Analytical Phase – Biological Factors
▪ NUTRITION
▪ In general, one night fasting (12 hours) is recommended in human medicine.
▪ The same can applies to animals. In particular, the
inhibition of postprandial lipemia may provide favorable results for clinical evaluation of many analytes.
▪ This can be done in some monogastric animals (cats and
dogs), and in ruminants it is applied or not (usually not).
Pre-Analytical Phase – Biological Factors
▪ NUTRITION
▪ In dogs, fasting urea or triglyceride concentrations may be lower than postprandial levels.
▪ With postprandial effect, levels of analytes such as serum concentration of glucose, pancreatic enzymes, bile acids and insulin vary.
▪ In newborns, serum total protein, immunoglobulin levels, ALP and GGT activities are significantly higher than
adults. This is due to colostrum ingredient.
Pre-Analytical Phase – Biological Factors
▪ STRESS
▪ Stress generally refers to two things: the psychological perception of pressure, on the one hand, and the body's response to it, on the other, which involves multiple
systems, from metabolism to muscles to memory.
▪ Through hormonal signaling, the perception of danger sets off an automatic response system, known as the
fight-or-flight response, that prepares all animals to meet
a challenge or flee from it.
Pre-Analytical Phase – Biological Factors
▪ STRESS
▪ In acute conditions, adrenal medulla is activeted. In subacute and chronic conditions adrenal cortex is activated.
▪ Depending on individual differences or species, factors such as transportation, discipline and environment are a source of stress.
▪ For example blood collection under stress conditions in
cats can cause hyperglycemia (up to 450 mg /dL) and
lymphocytosis.
Pre-Analytical Phase – Biological Factors
▪ STRESS
▪ Depending on the transport stress in dogs, ALP activity may increase, and again, hyperglycemia may occur.
▪ Glucocorticoid levels are elevated and leukocytosis is
seen in cattles due to stress associated with physical
fatigue, thirst, malnutrition, and transport.
Pre-Analytical Phase – Biological Factors
▪ DRUGS
▪ There are a variety of drugs that affect all aspects of hemostasis. Drugs or its metabolites affect analytes.
▪ For example, adrenocorticoid stimulants cause elevation in ALP activity.
▪ In animals, the effects of glucocorticoids and
nonsteroidal anti-inflammatory drugs have been
investigated and both haematological (leucocytosis) and
biochemical changes (increased ALP activity) have been
reported.
Pre-Analytical Phase – Biological Factors
▪ BIOLOGICAL RHYTHMS
▪ Analytes may vary due to biological rhythms or hormonal cycles, including reproductive cycles.
▪ The circadian rhythms of a particular analyte may vary from species to species.
▪ While cortisol levels in humans, monkeys and rats are
higher in the morning, it is reported that, in some cases,
there are almost no changes in dogs accordint to some
studies. However, in some cases there may be changes
especially in dogs living in groups.
Pre-Analytical Phase – Biological Factors
▪ AGE and GENDER
▪ Changes in serum biochemistry can be observed due to age and sex-dependent physiological variations.
▪ It has been reported that some hematological and biochemical parameters in dogs have changed
significantly depending on age.
▪ Serum phosphorus concentration and Ca/P ratio is
different depending on sex.
Pre-Analytical Phase – Biological Factors
▪ EXERCISE/TRAINING
▪ Different effects can be seen depending on the type and intensity of physical effort.
▪ Hematologic and biochemical changes such as high
hematocrit, c-reactive protein (CRP), lactate, potassium and ammonia concentration have been observed in
racing horses and dogs that are regularly trained.
▪ Especially after intensive physical activity, animals should
be rested before collecting specimens.
Pre-Analytical Phase – Biological Factors
Factors affecting the pre-analytical process directly affect laboratory analysis.
It is impossible to completely control these factors.
The important thing is to keep these factor
under control or not to ignore them.
Analytical Phase
Method Selection (Analysis Methods), Method Validation or Verification, Quality Control, Analytical, Analytical Instruments and Working Principles
Analytical Phase
▪ This phase includes what is usually considered the
"actual" laboratory testing or the diagnostic
procedures, processes, and products that ultimately provide results.
▪ It includes preparation of analytical samples, preparation of instruments, preparation/control of
reagents/chemicals, calibrations, internal quality controls
(External quality controls?), Analysis follow-up, obtaining
the results, control (repetition if necessary) and expert
approval.
Analytical Phase
1. QUALITATIVE METHODS 2. QUANTITAVIE METHODS
i. Colorimetric/Photometric methods
• (metabolites and enzymes)
ii. Potentiometric methods
• (Mineral ions and gases)
iii. Immunological methods
• RIA, ELISA, IFA... (Hormones, vitamins etc.)
Analytical Phase
▪ Clinical biochemical analyzes according to the items to be examined
1. Quantification of metabolites
2. Determination of the activities of enzymes 3. Quantification of vitamins
4. Quantification of hormones
5. Dynamic tests
Analytical Phase
▪ Clinical biochemical analysis according to the used reagent
1. Performed with manually prepared reagents
2. Performed with ready to use reagents and test kits 3. Performed with dipsticks (eg. Urinanalysis)
▪ Clinical biochemical analysis according to
manual processing and automation requirement
1. Manual analyzes. Manually processed methods.
2. Analyzes with automated systems.
Analytical Phase
▪ Colorimetry-Photometry
▪ Color formation is directly related to substance concentration. In other words, the color that absorbs the light is related to the color of the material itself, that is, its concentration.
▪ Photometers, colorimeters and spectrophotometers work according to this principle.
▪ It is widely used, especially to analyze metabolites.
Analytical Phase
▪ Colorimetry is the techniques that is frequently used in biochemical investigations.
▪ This involves the quantitative estimation of colors. This
means that if you want to measure the quantity of a
substance in a mixture, you could use the technique of
colorimetry, by allowing the substance to bind with color
forming chromogens. The difference in color results in the
difference in the absorption of light, which is made use of
here in this technique called colorimetry.
Analytical Phase
▪ Photometry is the science of the measurement of light, in terms of its perceived brightness.
▪ The basic principle of this technology involves measurement of quantity of light absorbing analyte in a solution. This can only be however applied to solutions which follow the Beer Lambert’s law. Analytes which have the tendency to absorb light, when exposed to a beam of incident light, will absorb some. This results in reflection of a light of lower intensity.
The intensity of the reflected light is then considered
inversely proportional to the concentration of the analyte of
interest in the solution.
Analytical Phase
▪ Spectrophotometry is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution.
▪ The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. This
measurement can also be used to measure the amount of a known chemical substance. Spectrophotometry is one of the most useful methods of quantitative analysis in various
fields such as chemistry, physics, biochemistry, material and
chemical engineering and clinical applications.
Analytical Phase
▪While the instruments that separate and transmit part of the light beam on the
analyzed sample using filters are called colorimeters or photometers, the
instruments that make this selection through slits or prisms are called
spectrophotometers.
Analytical Phase
Source: Wiki
Beer-Lambert Law
The Beer-Lambert law relates the attenuation of light to the properties of the material through which the light is traveling.
Transmittance (T) = I/I
0Absorbance(A) = -log (T) A = εcl
εcl = -log (T)
ε: molar extinction coefficient (L/mol/cm) C: concentration (mol/L)
l: length of the light path in cm
Analytical Phase
Analytical Phase
Source: ChemWiki.UC.Davis
Analytical Phase
Analytical Phase
Analytical Phase
Spectrophotometer Animation: Youtube
Analytical Phase
▪ Potentiometric Methods
▪ Potentiometry is one of the methods of electroanalytical chemistry. It is usually
employed to find the concentration of a solute in solution.
▪ In potentiometric measurements, the potential between two electrodes is measured using a high impedance voltmeter.
▪ It is often used to measure mineral ions and
gases.
Analytical Phase
▪ ISE (ION-SELECTIVE ELECTRODE)
▪ Ion Selective Electrodes (ISE) are membrane electrodes that respond selectively to ions in the presence of others. These include probes that
measure specific ions and gases in solution.
Kaynak: AOB Lab
Analytical Phase
▪ELECTROPHORESIS
▪ Electrophoresis is a
separations technique that is based on the mobility of ions in an electric field.
Positively charged ions
migrate towards a negative electrode and negatively
charged ions migrate
toward a positive electrode.
Analitik Süreç
Kaneko JJ, Harvey JW, Bruss ML, 2008. Clinical Biochemistry of Domestic Animals, 6th edi. Academic Press-Elsevier
Analitik Süreç
Kaneko JJ, Harvey JW, Bruss ML, 2008. Clinical Biochemistry of Domestic Animals, 6th edi. Academic Press-Elsevier
Analytical Phase
▪CHROMATOGRAPHY
▪ Chromatography’ is an analytical technique commonly used for
separating a mixture of
chemical substances into its individual components, so that the individual
components can be
thoroughly analyzed. There are many types of
chromatography.
Paper Chromatography
HPLC
Analytical Phase
▪ Immunological Methods
▪ RIA, ELISA, FIA, ECLIA, IFA etc...
▪ It is mainly used for hormone and vitamin analyzes in clinical
laboratories.
Post-Analytical Phase
Evaluation Of Results, Reference Value, Reporting
Post-Analytical Phase
▪ It is the final phase of the laboratory process.
▪ This phase culminates in the production of a final
value, result, or in the case of histology, a diagnostic pathology report.
▪ The results are evaluated according to the quality control criterias, the unit of measurement and the reference values are checked, the analysis report is prepared, the last control is made and report is
delivered to animal owner or the veterinarian
References
▪ eClinPath: www.eclinpath.com
▪ Hooijberg E, Leidinger E, Freeman KP. An error management system in a veterinary clinical laboratory. J Vet Diagn Invest. 2012; 24: 458–468.
▪ Jashnani KD, Karwande A, Puranik G. Icteric donor plasma: To transfuse or to discard?.
Indian J Pathol Microbiol 2012;55:604-5
▪ Karagül H, Altıntaş A, Fidancı UR, Sel T, 2000. Klinik Biyokimya. Medisan, Ankara
▪ Kaneko JJ, Harvey JW, Bruss ML, 2008. Clinical Biochemistry of Domestic Animals, 6th edi. Academic Press-Elsevier
▪ Pineda MH, Dooley MP, 2003. McDonald’s Veterinary Endocrinology and Reproduction, 5th edi. Blackwell Publishing.
▪ Thrall MA, Weiser G, Allison RW, Campbell TW, 2012. Veterinary Hematology and Clinical Biochemistry, 2nd edi. Wiley-Blackwell
