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CLINICAL PATHOLOGY المرحلة الرابعة// CLINICAL CHEMESTRY احمد نعمان.م Clinical chemistry Chemical measurement of various constituents of body fluid especially blood plasma or serum .one test commonly not enough to diagnosis a disease condition ,so its often need group of tests that gathered to analyze results and must be combined with other laboratory procedures (full hematology , bacterial culture , serology -----) , history , and physical examination . the clinician must have complete knowledge of the normal values . Blood samples collection and handling : Blood Samples Obtained For Clinical Chemistry Should Be Taken: ①carefully to avoid and minimize trauma either physical or psychological ,excitement and apprehension or exercise of animal that may cause hyperadrenalized . ②avoid contaminating the blood or the animal patient . by use sterile equipment , skin must be cleansed and disinfectant , using plastic disposable syringes and needles (if available vacutainers with or without anticoagulants ) , the anticoagulant of choice ( heparin , oxalate , citrate ,or EDTA) . ③needle should be removed due to forcing blood through a small gauge needle may disrupt erythrocytes ,also over shaking in mixing blood . ④chemical analyses of blood should made as soon as practical (within one hour following collection ) , can refrigerated immediately . the serum should be separated quickly as possible , coagulate at room temperature for max 30 minutes and clot gently removed from the sides of the tube by using wooden stick . then centrifuged and collected into clean tubes .plasma can be immediately separated by centrifugation ( care be taken not to over centrifuge the blood or at high speed ). Blood Samples Acceptable For Clinical Chemistry If Following Precaution Are Taken: 1- Avoid hemolysis by: a- Care in collection as in ①②. b- Carful transfer of blood as in ③. c- Separation of the blood as in ④. 2- Avoid volume changes due to dilution or evaporation . a- Avoid wet syringe . b- If possible not use liquid anticoagulant . c- Open container . 3- Avoid composition changes : a- Bacterial alteration . by ②④. b- Enzymatic alteration . by ②④. c- Blood gas changes .( reduced by filling the chamber nearly to capacity and keeping it tightly closed). d- Cell liquid interchanges .by ③④. 1 Coles , Embert H. (1986) "Veterinary clinical pathology" 4th ed. W.B .Sauenders CLINICAL PATHOLOGY المرحلة الرابعة// CLINICAL CHEMESTRY احمد نعمان.م All samples submitted must record owner's name , the case or accession number , date, specimen number , patient identification ,and test required . First must use best grade volumetric glassware ( national bureau of standards : including pipettes , flasks , burets ,and graduate cylinders ), special kits (developed for animal, not outdate , good storage) , and standardized capillary micropipette . Instruments : Must calibrated every time to diagnosis incompletely accuracy or problems by Pre calibrated scale Provide by manufacturing of test kit or instrument standard solution . Colorimetry : Its quantitative analysis in clinical chemistry . by converted body fluid ( such as blood) into colored solution by adding chemical reagents . then estimate intensity of color and compared to standards and by adding dilution until reaching similar color ( by photoelectric colorimetry that measure transmission light (%T) or absorbance light ). When reach similar color measure volume of diluents . Spectrophotometer : Widely used . by fixing desired wave length by using filters between 350-1000 nm. .after adding reagents measured difference of transmittance or absorbance light between sample and standard solution . Flame Photometry : Same Spectrophotometer but solution have metal ion released light when burned . Atomic Absorption Spectrophotometer : Same Flame photometry but it will measured light absorbed by ground state atoms rather than the light emitted by atoms . Multiple Channel Auto Analyzer Reflotron : a dry reagent method , it's using already added reagent strips that exactly calculated concentration , and measured sample kinetically . Enzymology Enzymes is protein products inside tissue cells act specific action of energy and different in each tissue . its present in blood due to 1- leakage (as result of cell damage ). 2-induced (when increased cell production due to several effects ). 2 Coles , Embert H. (1986) "Veterinary clinical pathology" 4th ed. W.B .Sauenders CLINICAL PATHOLOGY المرحلة الرابعة// CLINICAL CHEMESTRY احمد نعمان.م Kidney Function The kidneys are the chief organs regulating the internal environment of the body . Urine is a byproduct of these regulatory activates .(maintaining extracellular, and lesser intracellular fluid). involved : 12345- Elimination of water formed or introduced to body at normal amount required . Eliminate of inorganic elements according to the need of the body. Eliminate of non volatile end products of metabolic activity . Eliminate of foreign toxic substances. Retention required substances ( amino acids , hormones , vitamins , plasma portions , glucose ----) . 6- Formation and execration of substances as hydrogen ions and ammonia . So it play important role in the regulation of water , electrolyte , and acid – base balance . the Functional unit is Nephron , which consists of 2 units : ❶ Glomerulus . ( Filtration unit ) . ❷Tubules which lined by epithelial cell (execration , reabsorbtion, and other ) urine. Analysis of this end product of kidney function will often reveal alterations typical of diseases of that organ , with addition may provide information of other physiologic processes in the body . 12345- URINALYSIS . RENAL FUNCTION TESTS . BACTERIAL CULTURE . RENAL BIOPSY. THE HEMOGRAM IN RENAL DISEASE. RENAL FUNCTION TESTS Renal function tests do not reveal the definitive cause of disease , wither the disease is in an acute or chronic stage . its more accurate done by periodic assessment. 3 Coles , Embert H. (1986) "Veterinary clinical pathology" 4th ed. W.B .Sauenders CLINICAL PATHOLOGY المرحلة الرابعة// CLINICAL CHEMESTRY احمد نعمان.م Measurements can be made of the renal blood flow , the glomerular filtration , and the renal tubule excretion and reabsroption . its done by : ① urine specific gravity (SG) . ② estimation of nonprotein nitrogen (NPN) levels in the blood . ③ studies of the ability of the kidney to execrate certain dyes . ④ test based on the clearance concept. ② estimation of nonprotein nitrogen (NPN) levels in the blood: Its nitrogen – containing components of serum or plasma that are not associated with protein . including : Urea , Creatinine , Uric Acid , Ammonia , Amino Acids , and Undetermined Nitrogen . ( when all increased together termed Azotemia the Urea , and Creatinine is most used in veterinary medicine ) Urea Nitrogen (UN) : Urea is formed in liver and represents end product of protein catabolism . its no useful function in the body , and almost excreted by kidneys. The glomerulus filters urea and 25-40% reabsorbed through tubules . ( when it sever increased termed Uremia ) Indication for testing : 1- when suspected decreased kidney function. 2- measuring peripheral perfusion of tissues in hypovolemic shock or blood pressure . 3- routine pre surgical laboratory screening . UN increased normally with dietary in protein . and when dietary protein. UN in case of: ❶ catabolic breakdown of the tissues as a consequence of Fever ,Trauma , Infection , or Toxemia . ❷ Hemorrhagic into gastro intestinal tract . ❸drug increase protein catabolism [ corticosteroids , thyroid compounds]. ❹drugs decrease protein anabolism [tetracycline ]. ❺anything that reduces the glomerular filtration rat (GFR). ❻decreased plasma water . Azotemia may be prerenal , renal , or post renal . prerenal when blood flow though kidney (also blood pressure) as in [ shock , dehydration , cardiac diseases , and hypoadrenocortictism ]. Postrenal as[ blocked urethra , rupture bladder ]. UN in case of : gross sepsis , hormonal effect , liver failure , or portosystemic shunt . 4 Coles , Embert H. (1986) "Veterinary clinical pathology" 4th ed. W.B .Sauenders CLINICAL PATHOLOGY المرحلة الرابعة// CLINICAL CHEMESTRY احمد نعمان.م Creatinine (Cr) Formed during muscle metabolism of creatin and phosphocreatin . also its excreted by The glomerular filtration and reabsorbed through tubules. Factors affect (Cr) similar to (UN) except : 1-criatinine not influenced by diet . 2- daily production from muscle is relatively constant . 3-not affected by catabolic factor [like fever ---]. Other blood chemistry determinations : Electrolytes : Potassium: hyperkalemia in oliguric , anuric . Sodium : in chronic renal diseases with polyurea and water loss Na to maintain body isotonicity. Chloride : follow Na. Bicarbonate : in advanced renal diseases . Phosphate :hypophosphatemia when GFR is reduced . Calcium:hypocalecmia in chronic renal diseases . Nonelectrolytes : Blood pH : acidosis in renal failure . Serum protein : total serum protein reflect state of hydration . hyperproteinemia in dehydration . renal diseases protein urea hypo albuminemia (with edema) due to osmotic pressure . ③Dye Excretion Tests studies of the ability of the kidney to execrate certain dyes : By injected phenolsulfonphthalein (PSP) that removed from plasma by tubular excretion . (Dogs) ④ Renal Clearance Tests test based on the clearance concept. By measuring clear blood of some constituents in one minute . its depend on 1- glomerular filtration . 2- filtration plus tubular excretion. 3- filtration plus tubular reabsorption. Azotemia = UN + Cr Urine S.G : Concentrated pre renal (Pcv ,Alb. ----)in [ ADH ,Ca ,steroids , or pyometra) Non Concentrated Renal history (weeks , weight loss ) chronic (Pcv ,Alb ) ( hours , good body condition ) Acute (Pcv ,Alb.) Urinalysis : protein = glomerular casts = tubular Chronic = interstitial casts , WBC, RBc ,bacteria = pelvis Na : K >27 primary poly uric renal failure <27 hypoadrenocortictism renal failure 5 Coles , Embert H. (1986) "Veterinary clinical pathology" 4th ed. W.B .Sauenders