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CLINICAL CHEMISTRY SPECIMEN COLLECTION & OTHER PRE-ANALYTICAL VARIABLES BY: DR. CATHERINE CABABA General Methods of Blood Collection SKIN PUNCTURE & HEEL STIC...
CLINICAL CHEMISTRY SPECIMEN COLLECTION & OTHER PRE-ANALYTICAL VARIABLES BY: DR. CATHERINE CABABA General Methods of Blood Collection SKIN PUNCTURE & HEEL STICK ARTERIAL PUNCTURE VENIPUNCTURE KIN PUNCTURE S Sites of Puncture: Earlobe Palmar surface of the fingers Plantar surface of the heel and big toe Indications: Pediatric infants Geriatric patients Adults w/ exterme obesity, thrombotic tendency and severe burns emolysis may occur in skin puncture for the ff H reasons: Brachial artery at the antecubital fossa 1. There is residual alcohol at the skin puncture site. 2. Patients have increased red blood cell fragility and high packed cell volume (e.g., newborns and infants). Dorsalis pedis artery in the foot ARTERIAL PUNCTURE Act of obtaining blood from an artery More difficult toAct of obtaining blood from an artery More difficult to perform because of inherent arterial pressure, difficulty in stopping the bleeding afterwards, and undesirable development of hematoma Sites of puncture: Radial artery at the wrist Brachial artery in the elbow Femoral artery in the groin Arterial Puncture Sites: Femoral artery just below the inguinal ligament ertain areas are to be avoided when choosing a C site: Extensive scars from burns and surgery - it is difficult to puncture the scar tissue and obtain a specimen. Upper extremity on the side of a previous mastectomy - test results may be affected because of lymphedema. Hematoma - may cause erroneous test results. If another site is not available, collect the specimen distal to the hematoma. Intravenous therapy (IV) / blood Venipuncture transfusions - fluid may dilute the specimen, Open System so collect from the opposite arm if possible. Closed System Otherwise, satisfactory samples may be drawn below the IV by following these procedures: ○ Turn off the IV for at least 2 minutes before venipuncture. ○ Apply the tourniquet below the IV site. Select a vein other than the one with the IV. ○ Perform the venipuncture. Draw 5 ml of blood and discard befor drawing the specimen tubes for testing. Lines - Drawing from an intravenous line may avoid a difficult venipuncture, but introduces problems. The line must be ENIPUNCTURE V flushed first. When using a syringe inserted Most frequent site – antecubital fossa: into the line, blood must be withdrawn Cephalic vein – on the upper forearm and on slowly to avoid hemolysis. the thumb side of the hand Edematous extremities - tissue fluid Basilic vein – on the lower forearm and on accumulation alters test results. the little finger side of the hand Median cubital vein Steps in Venipuncture: 1. Carefully look over requisition slips. Note any special instructions. 2. Identify the patient. Always ask the patient to state his/her full name. 3. Verify diet restrictions. Fasting or non-fasting specimen? Note any medication that may interfere with testing. 4. Assemble equipment 5. Reassure and position the patient. Never tell the patient it will not hurt. The patient should be seated in a blood-drawing chair or lying down. 6. Apply torniquet. Applied 3-4” above the Other Sites: intended site and should not be left in place for longer than 1 minute. 7. Select venipuncture site. Have the patient make fist, to make the vein more prominent. Feel the vein. 8. Cleanse the site w/ 70% alcohol, in a circular motion 9. Uncover the needle and inspect for imperfections. 10. Perform the venipuncture. 11. Fill tubes: order of draw.(for evacuated system) 12. Release tourniquet. 13. Withdrawal and needle disposal. 14. Label tubes. tubes will be necessary BEFORE you begin to draw blood, and determine the order of draw for the tubes. Releasing the tourniquet When the final tube is being drawn, release the tourniquet. Then remove the tube, and remove the needle. Applying pressure over the vein After the needle is removed from the vein, apply firm pressure over the site to achieve hemostasis. Applying bandage Apply a bandage to the area. Disposing needle into sharps Dispose of the needle into a sharps Patient identification container that is close by. The first step is always to identify the patient. Outpatient phlebotomy, as shown Labeling the specimens here, should take place with the patient Label the tubes, checking the requisition for seated. the proper identification. Equipment Here is the equipment for performing phlebotomy. Barrier protection for the Order of Draw phlebotomist consists of the latex gloves. Recommended when drawing multiple specimens from a single venipuncture Apply tourniquet and palpate for vein Purpose : to avoid possible test result error The tourniquet is applied and the due to cross contamination of tube additives phlebotomist palpates for a suitable vein for drawing blood. Sterilize the site The area of skin is cleaned with a disinfectant, here an alcohol swab. Insert needle The vein is anchored and the needle is inserted. Drawing the specimen The vacutainer tube is depressed into the needle to begin drawing blood. Additional vacutainer tubes can be utilized. Determine what tests are ordered and what Collection Tube Colors & Additives Complications in Blood Collection CLINICAL CHEMISTRY CARBOHYDRATES BY: DR. CATHERINE CABABA Overview of Carbohydrate Metabolism Carbohydrates are called carbohydrates because they are essentially hydrates of carbon (i.e. they are composed of carbon and water and have a composition of (CH2O)n. The major nutritional role of carbohydrates is to provide energy and digestible carbohydrates provide 4 kilocalories per gram. No single carbohydrate is essential, but carbohydrates do participate in many required functions in the body. Glucose Comes from food. ○ Carbohydrates are the main dietary source of glucose. Glucose levels Disaccharides ○ rise after meals for an hour or two by a few grams ○ usually lowest in the morning, before the first meal of the day. Transported via the bloodstream Complex carbohydrates ○ from the intestines or liver to body Oligosaccharides cells, Polysaccharides ○ Starch G lucose is the primary source of energy for ○ Glycogen the body's cells ○ Dietary fiber ○ fats and oils being primarily a Starch compact energy store. F ailure to maintain blood glucose in the normal range leads to conditions of persistently high (Hyperglycemia) or low (Hypoglycemia) blood sugar. 1 20 grams of glucose / day = 480 calories Simple Sugars - Major storage carbohydrate in higher plants A mylose – long straight glucose chains Adipose and liver (a1-4) ○ Glucose acetyl CoA Amylopectin – branched every 24-30 glc ○ Glucose to glycerol for triglyceride residues (a 1-6) synthesis Provides 80% of dietary calories in humans ○ Liver releases glucose for other worldwide tissues Nervous system Glycogen ○ Always use glucose except during extreme fasts Reproductive tract/mammary ○ Glucose required by fetus ○ Lactose major milk carbohydrate Red blood cells ○ No mitochondria ○ Oxidize glucose to lactate ○ Lactate returned to liver for Gluconeogenesis Regulation of Carbohydrate Metabolism ajor storage carbohydrate in animals M Insulin Long straight glucose chains (a1-4) Glucagon Branched every 4-8 glc residues (a 1-6) Epinephrine More branched than starch Cortisol Less osmotic pressure Growth Hormone Easily mobilized Thyroxine Somatostatin Carbohydrate Metabolism Glycogenesis Beta Cells Glycogenolysis Beta cells form the Islets of Langerhans in Glycolysis (Embden-Meyerhoff Pathway) the pancreas Gluconeogenesis Insulin is produced only by beta cells in the pancreas Carbohydrates Glucagon also produced in the pancreas Serve as primary source of energy in the cell (acts to increase blood glucose levels when Central to all metabolic processes they are below normal) Secretion of Insulin Primarily in response to increase blood glucose levels Secondarily ○ Neural stimuli ○ Taste, smell of food ○ Increased amino acids in blood ○ Increased fatty acids in blood Normal Release of Insulin Normal fasting blood glucose ranges from arbohydrate Metabolism/ Utilization- Tissue C 80-90 mg per 100 ml Specificity Stored insulin released first Muscle – cardiac and skeletal Synthesis of more insulin triggered ○ Oxidize glucose/produce and store glycogen ○ Breakdown glycogen (fasted state) ○ Shift to other fuels in fasting state (fatty acids) C SF glucose conc is approximately 60% of plasma glucose conc. Urine glucose– renal threshold = 160-180 mg/dl Glucose methods Glucose Oxidase Glucose + O2 + H2O --------------gluconic acid + H2O2 2O2 + reduced chromogen-----------oxidized H chromogen + H2O Insulin affects many organs: alse low results: F It stimulates skeletal muscle fibers. →Inc. uric acid It stimulates liver cells. → Inc. bilirubin It acts on fat cells → Inc. ascorbic acid It inhibits production of certain enzymes. In each case, insulin triggers thes effects by Hexokinase binding to the insulin receptor. Glucose + ATP --------------------- G-6-P + ADP alse low results: F →Hemolysis →Inc. bilirubin Non Enzymatic Methods 1. Nelson- Somogyi – Copper reduction mtd Measure of true glucose Uses BaSO4 to remove non glucose reducing subs (saccharoids) Reagent: arsenomolybdic acid Product: aresenomolybdenum blue Conc of glucose is proportional to the absorbance of the solution Glucose Regulation by Insulin Liver 2. H agedorn-Jensen – Ferric reduction mtd ○ Gluconeogenesis Inverse colorimetry ○ Glycogenolysis Reagent: Ferricyanide (yellow) Muscle & adipose tissue Product: Ferrocyanide ( colorless) ○ Increased rate of glucose uptake4 Glucose conc. is proportional to the decrease Lipid metabolism in absorbance ○ Increased synthesis of lipids in liver and fat cells 3. O rtho-Toluidine ○ Attenuation of fatty acid release Condensation with aromatic amines from triglycerides stored in muscle Most specific among the non-enzymatic & fat tissue methods Reagent: O-toluidine in glacial acetic acid Product: Shiff’s base (green) eference Range R Conventional :70-110 mg/dl Conversion factor: 0.055 S.I : 3.9-6.1 mmol/L Genetic Defects in Carbohydrate Metabolism 1.Von Gierke’s dse Congenital form of glycogen storage dse Hypoglycemia Glucose Measurement Deficiency of the ENZ G-6-Phosphatase Specimens: whole blood, serum, plasma, urine, CSF 2. Galactosemia Whole blood glucose is lower by 15% Deficiency of galactose -1-phosphate uridyl compared with serum or plasma transferase Capillary blood sample is slightly higher compared with venous blood Disorder of Carbohydrate Metabolism iabetes Mellitus D Hyperglycemia (problem with glucose metabolism) Hypoglycemia Major health problem US/worldwide DM 3 Classical signs: ○ Polydipsia, ○ Polyphagia, ○ Polyuria DIABETES MELLITUS Complications [lousy blood vessels] isorders of Carbohydrate Metabolism D 1. Nephropathy Hyperglycemia 2. Neuropathy When blood glucose becomes high 3. Retinopathy - INSULIN allows glucose to enter cells 4. Atherosclerosis Liver - Production /storage of glycogen - lindness B - Inhibits glycogen breakdown - Renal failure - Increased protein & fat synthesis - Amputations (VLDL formation) - [heart attacks and strokes] Muscles - [OB/neonatal complications] - Promotes protein and glycogen synthesis he good news:Blood glucose control reduces T Fat cells complications of Diabetes! - Promotes storage of triglycerides Drowsy iabetes Mellitus : D Thirsty →a group of diseases characterized by high levelsof blood glucose resulting from defects in insulin ypoglycemia H production, insulin action, or both Diagnosed based on Whipple’s triad: 20.8 million in US ( 7% of population) Low blood glucose estimated 14.6 million diagnosed (only 2/3) Accompanied by typical symptoms Consists of 3 types: Resolved by glucose administration 1) Type 1 diabetes Weak, sweat 2) Type 2 diabetes Confused/irritable/ disoriented 3) Gestational diabetes Decreased plasma glucose levels Can be transient & relatively insignificant or Complications : life-threatening - Stroke Occurs in healthy-appearing and sick - Heart attack patients, as a result of reaction to medication - Kidney disease or of illness - Eye Disease Symptoms appear at a glucose level of about - Nerve Damage 50–55 mg/dL. Symptoms:increased hunger, sweating, ajor risk factors M nausea & vomiting, dizziness, nervousness - Family history & shaking, blurred speech & sight, mental - Obesity confusion - Origin (Afro-American, Hispanic, Native Glucagon: causes release of glucose from American, Asian-American) liver - Age (older than 45) ○ “glycogenolysis (breakdown of - History of gestational diabetes glycogen to glucose) - High cholesterol ○ “glyconeogenesis of glucose not - Hypertension available Lipolysis (breakdown of fat) Proteolysis (breakdown of amino acids) Glucose Tolerance Test Confirmatory test for DM Patient’s response to a glucose load is tested by measuring blood glucose at specific time intervals Types of GTT A. OGTT Patient Preparation: Normal to high carbohydrate intake for 3 days before the test Fasting for at least 10 hours Avoid medications like salicylates, diuretics, anti-convulsants, oral contraceptives, and corticosteroids 3 days prior to test Glucose load: 50 grms – children 75 grms – adults 100 grms- pregnant women Procedure: Determine FBS Administer glucose load Determine blood glucose after 30 min, 1 Who needs insulin medicine? hour, 2 hour and 3 hours Type I (insulin dependent) diabetes patients whose body produces no insulin. . I VGTT B Type 2 diabetes patients that do not always 25g/dl glucose solution administered produce enough insulin. intravenously within 1-2 minutes Treatment Blood drawn before infusion and at subcutaneous injection 1,3,5,10,20,30,40,60 and 120 minutes Criteria for the Diagnosis of Diabetes Mellitus following the end of infusion Three methods of diagnosis (each must be confirmed by one of the others on a Fructosamine subsequent day) Index for long term plasma glucose control 1. Diabetes symptoms + random glucose (2-3 week period), indicating compliance level of ≥200 mg/dL and efficacy of DM therapy 2. A fasting plasma glucose of ≥126 mg/dL 3. An oral glucose tolerance test (OGTT) Glycated hemoglobin (HbA1c) w/ 2-hour postload (75-g glucose level) Index for long term plasma glucose control ≥200 mg/dL (2-3month period), indicating compliance and efficacy of DM therapy P atients with following criteria have Specimen: EDTA whole blood sample “pre-diabetes”: Glycosylated Hemoglobin/Hemoglobin A1c Fasting glucose of ≥100 mg/dL but 200 mg/dl signals diabetes Ketones - Produced by liver through metabolism of A.K.A.: Glycated Hemoglobin tests A1C fatty acids - Provide a ready energy source from stored * **80 to 90 mg per 100 ml, is the normal fasting lipids blood glucose concentration in humans and most - Increase with carbohydrate deprivation or mammals which is associated with very low levels of decreased carbohydrate use (diabetes, insulin secretion. starvation/fasting, high-fat diets) - Three ketone bodies - Acetone (2%) - Acetoacetic acid (20%) - 3-β-hydroxybutyric acid (78%) - pecimen requirement is fresh serum or S urine. Diabetes Mellitus Prevention of effects: combination approach - Increased exercisE - Decreases need for insulin - Reduce calorie intake - Improves insulin sensitivity - Weight reduction - Improves insulin action Triad of Treatment D iet Medication ○ Oral hypoglycemics ○ Insulins Exercise Some things to know… D iabetic foot care - Dry, cracked skin + poor circulation could = loss of a limb - For the most part nurses don’t trim the nails of diabetic clients. Refer to Podiatrist. Typical diabetic foot ulcer CLINICAL CHEMISTRY PROTEIN BY: DR. CATHERINE CABABA General Characteristics - ets proteins apart from pure carbohydrates S Molecular Size and lipids, which contain no nitrogen atoms - Macromolecules – 6000 to several millions for some structural proteins S ynthesis - Synthesized in the liver, except for the Structure immunoglobulins (plasma cells) - Continuous chain of carbon & nitrogen atoms joined together by peptide bonds D istributions and Catabolism between adjacent amino acids - Distribution:extravascular, intravascular compartments Protein Structures - Catabolism:hydrolyzed to amino acids, Primary Structure– presents a linear deaminated producing ammonia & ketoacids sequence of amino acids - Ammoniaconverted to urea, excreted in the urine S econdary Structure– twisted shape of - Keto Acidsoxidized by means of Kreb’s primary structure due to rotation of bonds cycle – converted to glucose or fat T ertiaryStructure– three-dimensional Nitrogen Balance structure that forms when the amino acid chain folds back on itself N egative Nitrogen Balance - When protein catabolism exceeds protein Q uaternary Structure– individual proteins anabolism or monomeric subunits that form more - Occurs in excessive tissue destruction such stable complexes as dimers, trimers, etc. as burns, wasting disease, continual high fevers or starvation P ositive Nitrogen Balance - When anabolism is greater than catabolism - Occurs during growth, pregnancy, and repair processes Classification of Proteins As to Composition: Simple proteins - contain peptide chains which upon General Characteristics hydrolysis yield only amino acids - E.g. Albumin, globulin, albuminoids, Charge & Isoelectric Point histones, protamines - p H at which a particular protein has a net C onjugated proteins charge equal to zero - Composed of protein(apoprotein) and a - At a pH below their pI, proteins carry a net non-protein moiety (prosthetic group) positive charge; above their pI they carry a - E.g. net negative charge - Chromoprotein –myoglobin, hemoglobin Double charge - Metalloprotein– ferritin, ceruloplasmin - mino group= positive A - Lipoprotein– HDL,LDL,VLDL - Carboxyl group = negative - Glycoprotein– haptoglobulin, orosomucoid I mmunogenicity - Nucleoprotein– RNA,DNA - Effective antigens because of their large As to Shape: molecular mass, content of tyrosine and Globular specificity by species - spherical in shape, with mobile & dynamic function Nitrogen Content - E.g. hormones, enzymes, hemoproteins F ibrous . F D ractionation by Electrophoresis - elongated in shape, for structural purposes With the use of barbital buffer (veronal) at - E.g. Keratin, collagen, elastin pH 8.6, all serum proteins become negatively charged and migrate towards the As to Solubility: anode and produce a pattern of separation on Albumin – soluble in water, insoluble in electrophoretogram organic solvents a. solid support media – agarose gel and Globulin – insoluble in water, soluble in cellulose acetate organic solvents b. After migration, stains may be used to locate Albuminoids – insoluble in most common and identify the separated fractions on the reagents sample, e.g. Ponceau S, Bromphenol blue, - E.g. Collagen, elastin, keratin, & other and Coomassie brilliant blue fibrous proteins c. Fractions are quantitated using a densitometer General Functions of Proteins d. Total proteins are separated as 5 distinct Tissue nutrition ( amino acids from proteins bands can be used for the production of energy by e. From fastest to slowest : albumin, means of the citric acid cycle) alpha-1-globulin, alpha-2-globulin,beta Distribution of water among the globulin,and gamma globulin compartments of the body As buffers, maintain pH As transport proteins Protect the body against infection through antibodies and the complement system Act as receptors for hormones As enzymes, hasten biological chemical reaction As clotting factors, aid in hemostasis Structural role e.g. Collagen – major fibrous element of skin, bone, tendon, cartilage, blood vessels, and teeth Pathologic Serum Protein Electrophoresis Laboratory tests of Proteins Patterns Hepatic Cirrhosis Total Protein Methods: - When liver function is sufficiently A. Kjeldahl diminished, protein synthesizing capacity is Acid digestion of protein w/ measurement of compromised and concentrations of albumin total nitrogen and proteins in the alpha and beta bands are Reference method decreased. An additional common finding is 2-step reaction: beta-gamma bridging due to increased IgA. ○ Kjeldahlization – conversion of nitrogen to ammonia Nitrogen H2SO4 ammonia ○ ammonia measurement: 1. Nessler’s rxn 2. Berthelot rxn . B B iuret Formation of violet-colored chelate between cupric ions and peptide bond Composition of biuret reagent: - Cupric ion – breaks the peptide bonds - Tartrate salt – keeps copper in solution - Potassium iodide – stabilized cupric ions (prevents reduction to cuprous ion . R C efractometry Measurement of refractive index which reflects the concentration of proteins Rapid and simple N ephrotic Syndrome -- A cute Inflammation - Renal disease involving the glomeruli is - Alpha-1 and alpha-2 bands are increased always associated with increased urinary during the inflammatory response from protein loss. When protein loss is greater increased hepatic synthesis of acute phase than 3-4 g/day, the protein synthesizing reactant proteins. capacity of the liver is exceeded and hypoproteinemia, accompanied by anasarca, develops to cause the nephrotic syndrome. The massive urine protein loss is due to increased permeability of glomeruli to protein. The permeability increase may be minimal so that only albumin and other smaller molecular weight proteins are selectively filtered (selective nephrosis, as in Minimal Change Disease) or may be greater so that larger proteins are also filtered (nonselective nephrosis, as in membranous glomerulonephritis) as is the case in the example shown. Alpha-2-macroglobulin is sufficiently large so that it is not filtered and increased synthesis (from the general hepatic protein synthesis) causes its accumulation. C hronic Inflammation - Immunoglobulin synthesis by antigen activated B lymphocytes transformed to plasma cells is demonstrated by the increased polyclonal gamma band. A lpha-1-Antitrypsin Deficiency -- - A genetic defect causes a deficiency of alpha-1-antitrypsin. The antiprotease deficiency results in a propensity to develop emphysema. Since alpha-1-antitrypsin is the major component of the alpha-1 band, deficiency is suggested by a reduced alpha-1 band I mmunoglobulin Deficiency -- - Deficient immunoglobulin synthesis is revealed by a markedly diminished gamma band. Affected individuals are prone to recurrent infection. Alterations in Serum Total Proteins Hyperproteinemia: - Dehydration - Monoclonal or polyclonal gammopathies H ypoproteinemia: M onoclonal Gammopathy - Increased Protein loss – Nephrotic - An unusually sharp band in the gamma syndrome, blood loss, burns region strongly suggests the presence of a - Increased catabolism – homogeneous immunoglobulin and, thus, inflammation,pregnancy the malignant proliferation of plasma cells - Decreased synthesis – from a single cell (multiple myeloma) in liver disease,decreased amino acid intake contrast to the broad, heterogeneous, or polyclonal, gamma band as exhibited above Determination of Individual proteins in chronic inflammation from Albumin immunoglobulin synthesis by many different - protein present in highest concentration in clones of plasma cells. the serum - Homogeneous immunoglobulins are also - Produced by the liver found in Waldenstrom's macroglobulinemia - Functions: regulation of normal osmotic (where the sharp gamma band is always pressure; general transport protein IgM). Alterations in Serum Albumin Hyperalbuminemia: ○ Dehydration Hypoalbuminemia: ○ Increased albumin loss– Nephrotic syndrome, blood loss, burns ○ Increased albumin catabolism– inflammation,pregnancy ○ Decreased albumin synthesis– liver disease,decreased amino acid intake Globulin ○ Decreased in inflammation Total globulins are measured by subtracting albumin from the total protein concentration Bence Jones Protein ○ An abnormal protein in the urine of patients with multiple myeloma ○ Demonstrated by heat and acetic acid method: Bence Jones protein precipitates when solution is heated at 60-65 deg Celcius, becomes Conversion factor: 10 soluble when heated at 100 deg Celcius and re-precipitates when Fibrinogen the solution cools to 60-65 deg Most abundant of the coagulation factors Celcius responsible for the formation of fibrin clot Found in plasma not in serum Cryoglobulins Methods of Determination: ○ Serum globulins that precipitate on A. Parfentjev mtd – fibrinogen is precipitated cooling and re-dissolve on warming w/ ammonium sulfate & sodium chloride from a citrated plasma. Dilution of the mixture is read spectrophotometrically B. Howe’s mtd – fibrinogen is precipitated w/ calcium chloride and assayed using Kjeldahl mtd. Reference Value: 200-400 mg/dl ( 2.0-4.2 g/L) Miscellaneous proteins Alpha1-antitrypsin ○ acute phase reactant, makes up 90% of alpha-1 region ○ Function: neutralize trypsin-like enzymes that can cause damage to structural proteins ○ Decrease may cause early onset of emphysema or infantile hepatitis Alpha1-fetoprotein ○ Principal fetal protein ○ No known adult function ○ Increased concentration in adult may indicate hepatocellular carcinoma Haptoglobulin ○ Binds and transports free hemoglobin ○ Decreased in intravascular hemolysis Ceruloplasmin ○ Copper transport protein ○ Decreased in Wilson’s disease ○ Increased in copper toxicity Alpha2-Macroglobulin ○ One of the largest protein in the plasma ○ Dramatic increase in nephrotic syndrome Transferrin ○ Transports iron ○ Increased in iron deficiency anemia CLINICAL CHEMISTRY LIPID PROFILE BY: DR. CATHERINE CABABA Lipid Panel; Coronary Risk Panel S torage form of lipid in man Provides insulation to vital organs elated Tests:Cholesterol Cholesterol; HDL-C R Transported in the blood by chylomicrons Cholesterol; HDL-C; LDL-C Cholesterol; HDL-C; and VLDL LDL-C; Triglycerides Cholesterol; HDL-C; LDL-C; Causes turbidity of serum after meals Triglycerides; VLDL-C Cholesterol; HDL-C; LDL-C; Triglycerides; VLDL-C; Cardiac Risk Sources: Assessment Exogenous Endogenous Lipid profile group of tests that are often ordered together Cholesterol to determine risk of coronary heart disease Steroid alcohol tests that have been shown to be good Exogenous indicators of whether someone is likely to Endogenous ( free chole – 30%; choesterol have a heart attack tests that have been ester – 70%) shown to be good indicators of whether Transported in the blood by HDL and LDL someone is likely to have a heart attack or Associated with development of coronary stroke caused by blockage of blood vessels heart disease, atherosclerosis, M.I. or hardening of the arteries (atherosclerosis) Phospholipid Includes: Most abundant lipid Total cholesterol Important component of the cell membrane High density lipoprotein cholesterol Forms: (HDL-C) — often called good cholesterol Lecithin (70%) Low density lipoprotein cholesterol ○ a.k.a. phosphatidylcholine (LDL-C) —often called bad cholesterol ○ lung surfactant Triglycerides Sphingomyelin ( 20%) Cephalin ( 10%) An extended profile may also include: Very low density lipoprotein cholesterol L/S ratio – measured to detect fetal lung maturity (VLDL-C) Specimen : amniotic fluid Non-HDL-C Ratio: >3.1:1 is good: 99% of the amount Adults: filtered ○ 1.5 L/24 h ○ oliguria < 400 mL ○ anuria < 100 mL ○ polyuria > 3000 mL Children: ○ 1.5 ml/Kg of b.w./1 hour rine volume depends on how much you drink and U sweat. In health it is closely matched to water balance by the hormone ADH or vasopressin, AVP. e define abnormally low urine volume as a 24 hour W How do you know it’s broken? volume less than 400 mL. This is known as oliguria. Decreased urine production Clinical symptoms patient is considered anuric when there is no or A Tests little urine, less than 100 mL/24 h. here is no absolute definition for polyuria as some T people can drink an awful lot and match it with a high urine output. If a patient has a urine volume greater than 3 litres per day and is not drinking then this is polyuria. I shall review the tests in the left column today. The measurement of urine protein is important in certain conditions, e.g.diabetes. The detection of substances such as red cells or glucose could be an early indicator of renal damage. Where can it break? Pre-renal Renal (intrarenal) Post-renal (obstruction) Causes of kidney functional disorders Pre-renal e.g. decreased intravascular volume Renal e.g. acute tubular necrosis Postrenal e.g. urethral obstruction Oliguria is a significant finding in a patient. An example is provided by Case 3 in the Chem Path tutorials. The traditional classification of causes is into prerenal, renal and postrenal. Usually the cause of the oliguria is obvious and can be appropriately managed. Tests of renal function glomerular filtration rate=GFR plasma creatinine= Pcr plasma urea-Purea urine volume= V urine urea- Uurea urine protein urine glucose hematuria osmolality I shall review the tests in the left column today. The measurement of urine protein is important in certain conditions, e.g.diabetes. The detection of substances such as red cells or glucose could be an early indicator of renal damage. enal Function Tests- R Urine volumes Plasma urea (BUN) = BUN (blood urea nitrogen) Urea: product of protein catabolism Synthesized by liver, majority excreted by kidney, partially reabsorbed in tubules Plasma concentration increases with decreased GFR Urea is easily measured. It has a wide reference range and the value increases after a meal. Its concentration is increased in many different conditions which makes it sensitive to the presence of disease but a non-specific test. Glomerular filtration rate Glomerular filtration= major physiologic responsibility of kidney, GFR used as index of overall excretory function Methods: ○ clearence of inulin, creatinine, EDTA and DTPA (=both derivates of acetic acid), cystatin C ○ GFR= Ux x V (V=volum of urine/ 1 minute or 1 second) P x x= clearence of substance used Urea is easily measured. It has a wide reference range and the value increases after a meal. Its concentration is increased in many different conditions which makes it sensitive to the presence of disease but a non-specific test. C reatine: main storage compound of high energy phosphate needed for muscle metabolism. Creatinine: anhydride of creatine! I n most circumstances the measurement of plasma creatinine can provide a specific test of glomerular function. The reference range is wide. A body builder may have a plasma creatinine at the top end and an old lady a value at the low end and this reflects muscle mass. Plasma creatinine should not be measured until 8 hours after a meal as there is some evidence that the concentration increases after meat ingestion. lasma creatinine concentration increases when P GFR falls. The problem is that GFR has to fall quite a bit before plasma creatinine concentration reliably increases. here are some important analytical interferences T which you should check with the laboratory. A patient with ketoacidosis, jaundice or infection might nzymatic conductivity rate method for E have agents in the plasma which could invalidate the measuring urea measurement of creatinine. Overhead 1 follows J affe´ reaction for measuring creatinine, simple, but better is enzymatic method Creatinine + alkaline picrate solution Bright orange/red colored complex absorbs light at 485nm n early test to detect renal damage, for instance a A FR is not often measured in clinical practice. It G simple strip test for haematuria is important in requires a patient to come to hospital. Currently screening for heavy metal poisoning. people who are considering donating a kidney whilst they are alive have their GFR measured. Before here is a clinical need to monitor a patient with T administering a drug with potentially toxic effects renal disease and this is achieved by serial plasma some patients will require a GFR measurement before measurements. the chemotherapy. This enables the oncologist to We need to know when to start dialysis in renal calculate the exact dose of drug after estimating its failure and laboratory tests assist the clinical elimination rate. decision making. There are about a million nephrons in each kidney FR used to be measured by calculating the G and this represents a considerable functional reserve. clearance of inulin. Nowadays radioactive In renal disease about half the nephrons have to lose substances are used, either technetium labelled their functioning before the abnormality can be diethylenediaminetetra acetic acid DTPA or detected by conventional laboratory tests. 51-chromium labelled EDTA ethylenediaminetetra acetic acid. Urea in patients with kidney diseases Useful test but must be interpreted with Analytical methods (Cr) great care, urea plasma level is more than Normal range Pcr creatinine dependent on protein intake ○ Male 0.6-1.2 mg/dL, Most useful when considered along with ○ Female 0.5-1.0 mg/dL creatinine High in high protein intake, low in severe liver dysfunction Plasma creatinine and renal functions X -linked recessive disease Lack of HPRT causes a build-up of uric acid in all body fluids, and leads to symptoms such as severe gout, poor muscle control, and moderate retardation, which appear in the first year of life. Osmolality of urine Measures urine concentrating ability Depends on # of particles, not size or charge Largely due to ADH (antidiuretic hormone) Can reach maximum of 1200 mOsm/L Normal range: 300-900mOsm/L, plasma 285+10 Prior to collection, fluid intake restricted, first void submitted for evaluation Urine dipsticks Strip impregnated with reagents for the substances in question within a urine sample Substance level can be altered in the setting of pathology within the urinary tract Measured substances: Modern dipsticks with multiplied zones: ○ Protein, hemoglobin, glucose, urobilinogen, nitrite, leukocytes, specific gravity, and pH Should be a tool everywhere on the level of primary care!!! Lesch Nyhan Syndrome rare, inherited disorder caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) CLINICAL CHEMISTRY LIVER FUNCTION TESTS BY: DR. CATHERINE CABABA Anatomy of the Liver ( fibrinogen), II (prothrombin), V, VII, IX, XSynthesis of plasma proteins, such as albumin and globulin, and coagulation factors I (fibrinogen), II (prothrombin), V, VII, IX, X and XI, Blood detoxification and purification Carbohydrate Metabolism Through the process of glycogenesis, glucose, fructose, and galactose are converted to glycogen and stored in the liver. Through the process of glycogenolysis, the liver breaks down stored glycogen to maintain blood glucose levels when there is a decrease in carbohydrate intake. Through the process of gluconeogenesis, the largest internal organ in the body liver synthesizes glucose from proteins or weighs about 3 pounds fats to maintain blood glucose levels. Covered by Glisson's capsule divided into a large right lobe and a smaller Lipid Metabolism left lobe. Cholesterol synthesis hepatic lobules are the functioning units of Lipogenesis, the production of triglycerides the liver (fats). Each of the approximately 1 million lobules consists of a hexagonal row of hepatic cells Other functions called hepatocytes immunological effects- the Between each row of hepatocytes are small reticuloendothelial system of the liver cavities called sinusoids contains many immunologically active cells, Each sinusoid is lined with Kupffer cells, acting as a 'sieve' for antigens carried to it phagocytic cells that remove amino acids, via the portal system nutrients, sugar, old red blood cells, bacteria Synthesis of angiotensinogenSynthesis of and debris from the blood angiotensinogen, a hormone that is responsible for raising the blood Physiology of the Liver pressureSynthesis of angiotensinogen, a Bile production and excretion hormone that is responsible for raising the Excretion of bilirubin, cholesterol, blood pressure when activated by hormones, and drugs reninSynthesis of angiotensinogen, a Metabolism of fats, proteins, and hormone that is responsible for raising the carbohydrates blood pressure when activated by renin, a Enzyme activation kidney enzyme that is released when the Storage of glycogen, vitamins, and minerals juxtaglomerular apparatusSynthesis of Synthesis of plasma proteins, such as angiotensinogen, a hormone that is albumin and globulin, and coagulation responsible for raising the blood pressure factorsSynthesis of plasma proteins, such as when activated by renin, a kidney enzyme albumin and globulin, and coagulation that is released when the juxtaglomerular factors ISynthesis of plasma proteins, such apparatus senses low blood pressure as albumin and globulin, and coagulation factors I (fibrinogen), IISynthesis of plasma Breakdown proteins, such as albumin and globulin, and breakdown of insulinbreakdown of insulin coagulation factors I (fibrinogen), II and other hormones (prothrombin), VSynthesis of plasma breakdown of hemoglobinbreakdown of proteins, such as albumin and globulin, and hemoglobin, creating metabolitesbreakdown coagulation factors I (fibrinogen), II of hemoglobin, creating metabolites that are (prothrombin), V, VIISynthesis of plasma added to bilebreakdown of hemoglobin, proteins, such as albumin and globulin, and creating metabolites that are added to bile as coagulation factors I (fibrinogen), II pigment (bilirubinbreakdown of (prothrombin), V, VII, IXSynthesis of hemoglobin, creating metabolites that are plasma proteins, such as albumin and added to bile as pigment (bilirubin and globulin, and coagulation factors I biliverdin) b reakdown or modification of Direct-reacting Bilirubuin (conjugated) toxicbreakdown or modification of toxic elevations typically result from obstruction substances (eg. methylation) and most either within the liver (intrahepatic) or a medicinal products in a process called drug source outside the liver metabolism e.g. gallstones or a tumor blocking the bile conversion of ammoniaconversion of ducts ammonia to urea Indirect-reacting Bilirubin (unconjugated) Bilirubin elevated levels are usually caused by liver bile pigment produced by the breakdown of cell dysfunction haem and reduction of biliverdin e.g. hepatitis Unconjugated bilirubin is insoluble in plasma unless bound to protein, mainly albumin. S alicylates, sulphonamides, non-esterified fatty acids and reduced pH levels result in decreased protein-binding of unconjugated bilirubin. Normally, 95% of the circulating bilirubin is unconjugated. The bilirubin-albumin complex is dissociated by receptors on hepatocytes. The albumin remains in the plasma. The bilirubin is taken into the hepatocyte and conjugated by the enzyme bilirubin ongenital & Acquired Disorders of Bilirubin C UDP-glucuronyl transferase to form Metabolism bilirubin diglucuronide. It is this 1. Gilbert’s syndrome (Bilirubin Transpot water-soluble glucuronate derivative which Deficit) is excreted into the biliary system. - Impaired Cellular Uptake of Bilirubin In the gut, principally the colon, bilirubin glucoronides are degraded by bacteria and 2. C rigler Najjar Syndrome (Conjugation converted into a mixture of compounds, Deficit) known as urobilinogen or stercobilinogen; - No conjugation of Bilirubin these are water soluble. - Deficiency of UDP-GT Most of the urobilinogen is excreted in the - Leads to kernicterus – deposition of faeces where it is oxidised to urobilin which bilirubin in the brain is brown. Some is reabsorbed into the liver where it is re-excreted. When the amount of 3. D ubin-Johnson syndrome (Excretion urobilinogen is increased, some passes into Deficits) the systemic circulation and is excreted in - defective excretion of bilirubin by the liver the urine cells ypes of Bilirubin T Liver Function Tests “Conjugated" or "Direct bilirubin“ 1. Alanine transaminase (ALT) Water Soluble – Yes(bound to glucuronic Serum Glutamic Pyruvate Transaminase acid) (SGPT) or Alanine aminotransferase Reacts quickly when diazo reagent is added (ALAT) to the blood specimen to produce present in hepatocytes (liver cells) When a azobilirubin "Direct bilirubin"
