Clinical Chemistry 1 Laboratory Midterm - Glucose Determination PDF

CLINICAL CHEMISTRY 1 LABORATORY: MIDTERM LESSON 5: Glucose Determination Glucose Oxidase Results: Method values are derived by comparing the absorbance Summary and Principle...

CLINICAL CHEMISTRY 1 LABORATORY: MIDTERM LESSON 5: Glucose Determination Glucose Oxidase Results: Method values are derived by comparing the absorbance Summary and Principle of the unknown (U) with that of a standard (S) The accurate estimation of glucose is important in identically treated. the diagnosis and management of hyperglycemia Glucose (mg/dl) = Au/Asx100 and hypoglycemia. hyperglycemia may occur as a o where Au and as are the absorbances of result of diabetes mellitus, in patient receiving unknown and standard, respectively, and intravenous glucose fluids, or during severe stress. 100 the concentration of standard Hypoglycemia my be the result of an insulinoma, (mg/dl). insulin administration, inborn error of Example: Au= 0.370, As= 0.280 carbohydrate metabolism or fasting. measurement o Glucose (mg/dl) = 0.370/0.280 x 100= of blood glucose levels was among the first 132 chemical procedures employed in clinical Expected Values: medicine. Serum/Plasma= 70-105mg/dL The glucose oxidase methodology was introduced CSF= 40-75 md/dL by Keilin and Hartree in 1948. Keyston later these ranges shoud serve only as a guideline. it is reported use of the combined glucose oxidase- ultimately the responsibility of the laboratory to peroxidase reagent, followed by the Teller addition establish its own range of expected values, since of a chromogenic reagent to Keyston's procedure. differences exist between instruments, The Stanbio single reagent glucose method is laboratories, and local populations. based on a technique described by Trinder et. al. FBS Glucose is oxidized in the presence of glucose evaluation of blood sugar test oxidase (GODase). The hydrogen peroxide formed types od blood test reacts, under the influence of peroxidase (POD), fasting: 8 hours with phenol and 4-aminoantipyrine to form a red- postprandial test: eating after fasting and the vioet quinone complex. the intensity of the color is proceed for blood test proportional to glucose concentration. blood sugar (Random): blood collected at any given time β-D- Glucose+H20+02--> GODase-->H202 +D-gluconic acid hbA1c: (glycated hemoglobin test)- precise blood H202 + 4-aminoantipyrine+phenol--> POD--> Quinone sugar detection for 3 months. Preferred by most of complex + H20 physicians. Reagents: Why we do testing: Buffer For diagnosis of diabetes Phenol Family history of family diseases 4-aminoantipyrine Patient gas associated diseases. glucose oxidase peroxidas LESSON 6: Glucose Determination: Dubowsky Method by non reactive ingredients Profame Preservatives GLUCOSE DETERMINATION it is supplied ready to use. Glucose is a simple sugar containing 6 carbon Glucose Standard: atoms. Glucose is an important source of energy in contiains 5.55mmol/L Glucose in the body and the sole source of energy for the 0.5 mol/L benzoic acid brain. Glucose is stored in the body in the form of Reagent Storage and Stability: glycogen; the concentration of glucose in the blood Reagent @ 2-8*c is maintained at around 5mmol/L by a variety of Standard @ 2-8*c hormones, principally insulin and glucagon. If the Glucose @ 2-8*c or @ -20*c blood glucose concentration falls below this level, Specimen Collection and Storage: neurological and other symptoms may result Non hemolyzed serum (hypoglycemia). Conversely, if the blood glucose Flouride level is raised above its normal level to 10mmol/L, Interfering substances: the condition of hyperglycemia develops. ascorbic acid This is a symptom of Diabetes Mellitus. Manual Procedure: METHOD 1. Zero spectrophotometer at 500 nm with distllied Method – Ortho-Toluidine, Dubowski Method water. Principle: Glucose condenses with O-toluidine in 2. For each Standard and sample tubes, add 1.0 ml glacia acetic acid when heated at 100*C forming N- reagent to test tubes and warm at 37*c for 5 glycosylamine minutes. 3. 3. add 10 ul of each sample to its respective test Glucose + 0-toluidine--> N- tubes, mix gently and return to 37*c incubation. Glycosylamine 4. 4. after 5 minutes of incubation, read and record Specimen Collection and Preparation the absorbance of a samples MAALIHAN H. | BSMLS 3-Y1-3 1  CLINICAL CHEMISTRY 1 LABORATORY: MIDTERM Serum: Remove from the clot within 30 minutes of Mix reagent and sample for 10 mins at 37 degrees Celsius. collection in order to prevent glycolysis Plasma: An anticoagulant containing fluoride Is LESSON 7: LIPID DETERMINATIONS recommended but any of the common NEW TOTAL CHOLESTEROL DETERMINATION anticoagulants may be used if plasma is separated Cholesterol is a waxy material which forms plate- from cells promptly after centrifugation like crystals found in blood, bile and brain. It acts Spectrophotometer as a precursor of bile acids and steroid hormone. Accurate pipetting devices The main interest in cholesterol comes from the Heating block or water bath (100*C degrees relationship between cholesterol and Celsius) atherosclerosis Cuvets Atherosclerosis ultimately results in the Interval timer deprivation of oxygen and blood to the tissues Reagent kits which are the underlying factor in most coronary Glucose reagent heart disease and stroke. o Gluco-Toluidine Reagent The enzymatic methods for quantitation of serum o Acetic Acid cholesterol demonstrate greater reliability and Standard accuracy but these approaches are more costly o Glucose Standard, 100 mg/Dl than the chemical methods Reagent Storage and Stability Method – Quantitative Enzymatic-Colorimetric the reagent is stable up to the end of its labeled Determination of Total expiration date, if properly stored at 2-8*C, Cholesterol in Serum or Plasma protected from light and contamination is avoided. Principle Do not freeze the reagent! Discard if it is found to Cholesterol esterase (CE) hydrolyzes esters to free contain particulate matter. the standard is stable cholesterol and fatty acids. The free cholesterol so up to the end of the labeled expiration date, if produced plus the preformed cholesterol are then properly stored at 2-8*C and contamination is oxidized in the presence of cholesterol oxidase avoided (COx) to cholest-4-en-3-one and hydrogen peroxide. Manual Procedure A quinoneimine chromogen, with absorption Pipet into cuvets the following volumes (mL) and maxima at 500nm, is produced when phenol is mix well oxidatively coupled with 4-aminophenazone in the presence of peroxidase (POD) with hydrogen peroxide. The intensity of final color is proportional to total cholesterol concentration. Lipid clearing factor (LCF): a mixture of special additives developed by Stanbio is integrated into the cholesterol reagent to help minimize Incubate all tubes at 100*C degrees Celsius for 3 interference due to lipemia. minutes. CE Immerse test tube in room temperature water for Cholesterol ester ----------> Cholesterol + Fatty acids 2 minutes to cool, read absorbance of each tube COx against Water Bak at 600 nm. Cholesterol + O2 -------> Cholesten-3-one + H2O2 Formula for glucose Concentration POD 2H2O2 + 4-aminophenazone + phenol ------> O- quinoneimine dye Specimen Collection and Preparation Normal values: (Profame) Blood should be collected following a 12-hour Serum/Plasma (Fasting): 65-110 mg/dL fast. Specimen may be serum, or plasma collected Spinal Fluid: 40-70 mg/dL with EDTA as an anticoagulant. Avoid hemolysis Clinical Significance: Materials Diagnosis if the patient has – Diabetes Mellitus- Spectrophotometer Disease characterized by insufficient secretion of Accurate pipetting devices insulin from the pancreas Heating block or water bath (37 degrees Celsius) Supplemented videos: Anamol laboratories Cuvets Cholesterol reagent – readily use Interval timer Cholesterol reagent of 200 mg Reagents 1000 ul / 10 ul pipettes 4-aminophenazone Reagent Standard Test Phenol Reagent 1000ul 1000ul 1000ul Peroxidase Standard n/a 10ul n/a Cholesterol esterase Sample 10ul Cholesterol oxidase MAALIHAN H. | BSMLS 3-Y1-3 2  CLINICAL CHEMISTRY 1 LABORATORY: MIDTERM Buffers and stabilizers MateriaIs: Cholesterol standard (200 mg/dL) Spectrophotometer o Buffered aqueous solution of cholesterol Accurate pipetting devices with stabilizers, surfactants and Heating block or water bath (37 degrees Celsius) preservative Cuvets Manual Procedure Vortex mixer 1. Pipet into cuvets the following volumes (mL) and Interval timer mix well Reagents: Enzymatic triglyceride reagent Triglyceride activator Triglyceride standard Preparation: 2. Incubate all cuvets at 37 degrees Celsius for 5 Add 9 drops of triglyceride activator to one bottle minutes. of triglyceride reagent, or add 50 μL of activator 3. Read S and U vs RB at 500nm within 60minutes. for every 5.0 mL of reagent. Invert gently 3 – 4 Formula for Cholesterol Concentration: times. Formula for Cholesterol Concentration: Before use allow to stand for at least 15minutes at C (mg/dL) = Asample/ Astandard x Value of standard room temperature. (200mg/dL) MANUAL PROCEDURE Reference Range: 1. Pipet into cuvets the following volumes (mL) and Adult: 140-310 mg/dL mix well: Chidren: 120-200 mg/Dl REAGENT STANDARD SAMPLE (U) NEW TRIGLYCERIDES DETERMINATION BLANK Triglycerides is a lipids or neutral fats consisting of REAGENT 1.0 1.0 1.0 glycerol combined with three fatty acids STANDARD 0.010 molecules, they are the main storage lipids in man SAMPLE 0..010 and constitute about 95% of adipose tissue lipids. NOTE: Volumes may be increased if the instrument They are found in the plasma as; part of the requires a volume greater than 1.0mL lipoprotein in various concentration. In general, 2. incubate all cuvets at 37 degree Celsius for 5 the higher the concentration of triglycerides, the minutes. lower the density of the lipoprotein. The main 3. Read S and U vs RB at 500 nm triglycerides carrying lipoprotein are the chylomicrons and VLDL (very low density Formula for Triglyceride Concentration lipoprotein). VLDL forms the great bulk of plasma C (mg/dL ) = Asample / Astandard X Value of standard triglyceride in post absorptive state. (200mg/dL) Method – Quantitative Enzymatic – Colorimetric Determination of Triglycerides in Serum or Plasma Normal Value: Principles TAG: Glycerol and fatty acids are first formed by lipase 67-157 mg/dI or 0.11-2.15 mmoI/I action on the triglycerides. TotaI Cholesterol: Glycerol is then phosphorylated by adenosine 5 140-200 up to 310 (Stanbio)mg/dI or 3.6-5.2 triphosphate (ATP) to produce glycerol – 3 mmoI/I phosphate (G-3-P) and adenosine 5 diphosphate HDI ChoIesteroI: (ADP) in a reaction catalyzed by glycerol kinase M: 29-60 mg/dI or 0.75-1.6 mmoI/I (GK) F: 38-75 mg/dI or 1.0-1.94 mmoI/I PREPARATION OF SOLUTIONS AND DILUTIONS UNITS OF MEASUREMENT TWO MAJOR SYSTEMS English System Metric System Specimen Collection and Preparation Sample stability: triglycerides are reportedly stable for at least 10 days at 2 – 8 degrees Celsius. Do not store samples at 15 – 25 degrees Celsius as phospholipids may hydrolyze, releasing free glycerol and falsely elevating triglyceride values International d’Unités MAALIHAN H. | BSMLS 3-Y1-3 3  CLINICAL CHEMISTRY 1 LABORATORY: MIDTERM Also known as le Systeme International in French, refers to the weight or volume of the solute or the SI Unit present in a specified amount of solvent or a Based on the Metric System solution THREE BASIC TYPES OF SOLUTIONS: o Percent solutions o Molar solutions o Normal solution A. PERCENT SOLUTIONS Amount of solute in a solution can be measured as a percentage of the total volume of the solution Expressed as equal parts per hundred or the amount of solute per 100 total units of solution Three expressions of percent solution Prefixes Used in the SI System Percent by mass is the mass of solute in a (mass-mass solution divided by the total percent or %w/w) mass of solution, multiplied by 100 (to put the value in terms of percentage). = mass of solute x 100 mass of solution Percent by volume is the volume of solute in a (volumevolume solution divided by the total percent or %v/v) volume of solution, multiplied by 100. = volume of solute x 100 volume REPORTING OF MEASUREMENTS of solution Components of a Laboratory Result: Mass-volume is the mass of solute in a solution o Actual value percent (%w/v) (in grams) divided by the total o Unit volume of solution (in milliliters), It is recommended that analytes be reported using multiplied by 100. moles of solute per volume of solution (substance = mass of solute (g) x 100 volume concentration) of solution (mL) Reporting laboratory results is often expressed in Practice Question: terms of substance concentration (e.g. moles) or 1. What is the percent-by-mass concentration of the mass of a substance (e.g. mg/dL, g/L, mEq/L sucrose in a solution made by dissolving and IU) rather than SI units Given: SOLUTIONS 7.5g sucrose Matter 86.5g water 2 types: 1. Pure substances o Elements o Compounds 2. Mixtures a. Homogenous 2. What is the percent-by-volume concentration if a b. Heterogenous 2mL of concentrated HCl is diluted with 80mL homogenous mixture of two or more substances with each distilled water? substance retaining its own Given: 2mL conc. HCl 80mL dH2O chemical identity A solution contains two or more components: a solvent and one or more solutes. Solutions used in laboratories and clinical settings are most often liquids, and the solvent is nearly always water. 3. What is the concentration of a 200 mL solution Solvents: components of solution that is present containing 1.8g of NaCl? in the greatest amount Given: 1.8g NaCl 200mL solution Solute: component of a solution that is present in a lesser amount relative to that of the solvent. CONCENTRATION OF SOLUTIONS MAALIHAN H. | BSMLS 3-Y1-3 4  CLINICAL CHEMISTRY 1 LABORATORY: MIDTERM mol of solute M=L of solution g of solute M=MW x L of soln’ CALCULATING THE AMOUNT OF SOLUTE OR SOLVENT IN A 1. Determine the molarity of a solution containing GIVEN PERCENT SOLUTION 4.35 moles of KMnO4 dissolved in enough water to 4. Normal saline solution (NSS) is used to dissolve give 750 mL solution drugs for IV use which is 0.9% w/v NaCl in water. Given: How many grams of NaCl is needed to prepare a 4.35 moles KMnO4 35mL NSS? 750mL solution Given: Volume of solution = 50mL Mass-volume percent = 0.9% NaCl solution (NSS) 2. Determine the molarity of a solution containing 20g NaOH dissolved in enough water to give 1.50L solution. (Na-23; O-16, H-1) Given: 20g NaOH 1.50L solution 5. 10% bleach (sodium hypochlorite) is used to disinfect benches before and after work. It denatures protein in micro-organisms and is 3. How many grams of FeSO4 is needed to prepare therefore effective in killing bacteria, fungus and 0.3L of 0.10M FeSO4 solution? (Fe-55.85; S-32.06; viruses. How much bleach is needed to make O-14) 100mL of 10% bleach (sodium hypochlorite) solution? How much distilled water is needed to Given: dilute the bleach? 0.3L solution Given: 0.10M Volume of solution = 100mL Percent by volume = 10% bleach (sodium hypochlorite) C. NORMAL SOLUTIONS least likely to be encountered of the four concentration expressions to be encountered in the clinical laboratories, but is often used in chemical titrations and chemical reagent classification 6. 10% bleach (sodium hypochlorite) is used to The number of gram equivalent weight per 1 L of disinfect benches before and after work. It solution denatures protein in micro-organisms and is g of solute therefore effective in killing bacteria, fungus and M= EW X L of solution viruses. How much bleach is needed to make Equivalent Weight = MW x valence 100mL of 10% bleach (sodium hypochlorite) IDENTIFYING THE VALENCE OF ACIDS, BASES, AND SALTS solution? How much distilled water is needed to ACIDS – count the number of Hydrogen ions dilute the bleach? BASES – count the number of Hydroxide ions Given: SALTS – multiply the absolute value of the ions Volume of solution = 100mL Percent by volume = 10% bleach (sodium hypochlorite) Practice Question: 1. Determine the normality of a solution containing 15g KCl dissolved in enough water to give 0.20L MOLAR SOLUTIONS solution. (K-39.10; Cl-35.45) solution containing one gram molecular weight (one Given: mole of the solute in one liter solution) of the 15g KCl substance per liter of the solution 0.20L solution MAALIHAN H. | BSMLS 3-Y1-3 5  CLINICAL CHEMISTRY 1 LABORATORY: MIDTERM Relationship of Normality and Molarity Normality is ALWAYS equal or greater than molarity of that compound Practice Questions: What is the molarity of a 2N NaCl solution? What is the normality of a 5M H2SO4 solution? DILUTIONS represents the ratio of concentrated or stock material to the total final volume of a solution and consists of the volume or weight of the concentrate plus the volume of the diluent, with the concentration units remaining the same. In the molar, normal or percentage solutions, the amount of solute contained in a given volume of solution is equal to the product of volume times the concentration. Whenever the solution is diluted, the volume is increased and its concentration is decreased but the total amount of solute remain unchanged. C1V1 = C2V2 Practice Question: 1. What is the initial volume of a 40% formaldehyde diluted to prepare 100mL of 10% formaldehyde solution Given: V1 = ? C1 = 40% ; V2 = 100mL C2 = 10% 2. What is the final concentration of a 50mL 90% methanol diluted to prepare a 200mL methanol solution? ***yawkona MAALIHAN H. | BSMLS 3-Y1-3 6