(Clinical Biochemistry) -...

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Physiopathology (Clinical Biochemistry) Serkan SAYINER, DVM PhD. Assist. Prof. Near East University, Faculty of Veterinary Medicine Department of Biochemisty [email protected]

Transcript of (Clinical Biochemistry) -...

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Physiopathology (Clinical Biochemistry)Serkan SAYINER, DVM PhD. Assist. Prof.

Near East University, Faculty of Veterinary Medicine

Department of Biochemisty

[email protected]

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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)

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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

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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

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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.

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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.

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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.

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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.

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Biochemistry

and

Clinical

Biochemistry

Genetics

Molecular

Biology

Toxicology

ImmunologyMicrobiology

Virology

Endocrinology

Physiology

Pharmacology

Veterinary

Medicine

Human

Medicine

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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

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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

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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 ....

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Minerals

▪ Macro and Micro (trace) elements.

▪ They have important functions in body fluids and tissues such as energyproduction, 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

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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

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BiologicalMaterialsBlood, Urine, Stool, Lymph, Sweat, Tear, Digestive Secretions, Puncture Fluids, CSF, Genital Secretions, Stones And Tissues

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Blood

▪ It is a biological fluid circulating in the vascular system.

▪ Contents: Molecules in solute state + cellular components in suspended state

Source: FisherBioservices

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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.

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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?

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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?

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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 H2O are reabsorbed in the connective tubes(ADH?).

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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

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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

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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 beexamined.

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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.

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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).

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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.

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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.

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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.

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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.

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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.

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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.

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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 inaccurateamount of blood will affect test results. These are frequently encountered pre-analytical errors in clinical laboratories.

!

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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.

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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).

!

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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.

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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.

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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.

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Pre-Analytical Phase – Technical Factors

SYNOVIAL FLUID

▪ There may be differences in the chemical and cellular components of the synovial samples collected from differentjoint 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.

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Pre-Analytical Phase – Technical Factors

TEMPERATURE▪ Generally, samples are stable at room temperature

for 2 hours and analysis results can be determinedat optimal levels.

▪ For light sensitive analytes such as bilirubin, thesamples should be kept in the dark conditions.

▪ In hematological tests to be performed with wholeblood, samples should not be frozen.

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Pre-Analytical Phase – Technical Factors

TEMPERATURE

▪ Samples must be stored at –20 ºC if it is wanted to keepit 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 thelaboratory should always be freshly collected.

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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.

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Pre-Analytical Phase – Technical Factors

CENTRIFUGE

▪ RCF or RPM?

oRCF=1.118 x 10-5 x r x rmp2

▪ In cytological samples, a lower g force is used to increase cellconcentration and preserve cell morphology.

▪ Since there is no relationship between the number of cellsand crystals and speed or force in the urine samples, 400-3900 g x 5 minutes can be used.

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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 tothe release of hemoglobin from within the red blood cells intothe blood plasma. According to degree of hemolysis, colour of serum changes from light to dark red. Hemolysis is the mostfrequently observed error in the pre-analytical phase both in human and veterinary laboratories.

▪ Especially in spectrophotometric analyzes it causes false results.

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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

≥ 300AST, Cholinesterase, CK, Fe, LDH, K,

P

HCO3-, Direct Bilirubin, Bile Acids

GGT

≥ 400

AST, Cholinesterase, CK, Fe, LDH, K,

P, Mg, Total Protein, TriglyceridesHCO3

-, 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.

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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

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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.

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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, theicteric index should closely match the value seen.

Icteric Index False Increase False Decrease

≥ 9 Cholesterol, Triglycerides, Creatinine, Bile Acids

≥ 19Cholesterol, Triglycerides, Creatinine, Bile Acids,

Total Protein

≥ 40 MgCholesterol, Triglycerides, Creatinine, Bile Acids,

Total Protein, GGT, Lipase, ,Uric Acid

≥ 55 MgCholesterol, Triglycerides, Creatinine, Bile Acids,

Total Protein, Lipase, Uric Acid, Amylase

Note: There may be differences between species.

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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.

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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.

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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).

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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, potassiumand 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.

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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 factorunder control or not to ignore them.

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Analytical PhaseMethod Selection (Analysis Methods), Method Validation or Verification, Quality Control, Analytical, Analytical Instruments and Working Principles

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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, obtainingthe results, control (repetition if necessary) and expert approval.

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Analytical Phase

1. QUALITATIVE METHODS

2. QUANTITAVIE METHODSi. Colorimetric/Photometric methods

• (metabolites and enzymes)

ii. Potentiometric methods

• (Mineral ions and gases)

iii. Immunological methods

•RIA, ELISA, IFA..... (Hormones, vitamins etc.)

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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

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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.

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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.

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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.

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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.

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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.

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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.

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Analytical Phase

Source: Wiki

Beer-Lambert LawThe Beer-Lambert law relates the

attenuation of light to the properties

of the material through which the

light is traveling.

Transmittance (T) = I/I0Absorbance(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

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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.

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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.

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Analitik Süreç

Kaneko JJ, Harvey JW, Bruss ML, 2008. Clinical Biochemistry of Domestic Animals, 6th edi. Academic Press-Elsevier

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Analitik Süreç

Kaneko JJ, Harvey JW, Bruss ML, 2008. Clinical Biochemistry of Domestic Animals, 6th edi. Academic Press-Elsevier

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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

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Analytical Phase

▪ Immunological Methods▪ RIA, ELISA, FIA, ECLIA, IFA

etc...

▪ It is mainly used forhormone and vitamin analyzes in clinical laboratories.

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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

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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