Clinical Chemistry 1 Past Paper PDF

MLS 301 – CLINICAL CHEMISTRY 1 First Semester AY 2024-2025 Zharina Leih Panopio-Atienza | Loren Deduyo Instructors College of Allied Medical Professions Carbohydrates CLINICAL CHEMISTRY 1 LABORATORY OBJEC...

MLS 301 – CLINICAL CHEMISTRY 1 First Semester AY 2024-2025 Zharina Leih Panopio-Atienza | Loren Deduyo Instructors College of Allied Medical Professions Carbohydrates CLINICAL CHEMISTRY 1 LABORATORY OBJECTIVES: Describe the principle, specimen of choice, and the advantages and disadvantages of the glucose analysis method. Describe the methods of glycated hemoglobin, specimen, specimen of choice and source of error. Describe the use of glycated hemoglobin in the long-term monitoring of diabetes SPECIMEN CONSIDERATIONS Specimens for carbohydrate analysis: Whole blood Serous fluid Plasma Synovial fluid Serum CSF Urine* Standard clinical specimen: FASTING VENOUS PLASMA SPECIMEN CONSIDERATIONS Serum is appropriate for glucose analysis if it is separated from the cells immediately after centrifugation (approximately 30 minutes after blood collection) Bacteria, WBCs and RBCs might consume the glucose present in the sample can LOWER glucose results If processing will be delayed for more than 30 minutes, use SODIUM FLUORIDE Fluoride binds magnesium, which inhibits the enzyme ENOLASE necessary for glycolysis. Causes false negative BUN result. If whole blood is refrigerated, 2 mg of NaF/mL of whole blood prevents glycolysis for up to 48 hours SPECIMEN CONSIDERATIONS ▪ Fasting blood sugar should be obtained after 8-10 hours of fasting (but not >16hrs) ▪ CSF glucose concentration is approximately 60-70% that of plasma concentrations. ▪ Blood glucose should be obtained 1-2 hours BEFORE spinal tap. ▪ Peritoneal fluid glucose is the same as plasma glucose ▪ Whole blood gives approximately 10-15% LOWER glucose levels than serum or plasma. ▪ Venous blood glucose is 7mg/dL LOWER than capillary blood glucose due to tissue metabolism ▪ Capillary blood glucose is the same with arterial blood glucose. SPECIMEN CONSIDERATIONS Glucose is metabolized at room temperature at a rate of 7mg/dl/hr. At 4°C, glucose decreases by approximately 2mg/dl/hr.* The rate of metabolism is higher with bacterial contamination or leukocytosis. In serum specimens without bacterial contamination or leukocytosis, results are clinically acceptable up to 90 minutes before separation of serum from cells. GLUCOSE METHODOLOGIES I. CHEMICAL METHODS (oxidation-reduction methods) 1. Copper reduction A. Folin Wu Method B. Nelson Somogyi C. Neocuproine method D. Benedict’s method E. Clinitest Method 2. Ferric reduction (Hagedorn-Jensen) 3. Condensation method II. ENZYMATIC METHODS 1. Glucose Oxidase 2. Hexokinase-G6PD I. Chemical Methods 1. COPPER REDUCTION METHOD OLDEST METHOD Principle: Glucose and other reducing sugars convert cupric to cuprous ions in the presence of heat and alkali CuSO4 + Glucose heat ↓ alkali Cu2O + oxidized substance ↓ Color formation I. Chemical Methods 1. COPPER REDUCTION METHOD A. FOLIN WU METHOD SENSITIVE but not specific MAJOR DISADVANTAGE: non-glucose reducing substance also reacts with the test Cuprous ions + Phosphomolybdate ↓ Phosphomolybdenum BLUE I. Chemical Methods 1. COPPER REDUCTION METHOD B. NELSON SOMOGYI METHOD SENSITIVE and SPECIFIC After PFF preparation, non-glucose reducing substances are adsorbed by barium sulfate Cuprous ions + Arsenomolybdate ↓ Arsenomolybdenum BLUE I. Chemical Methods 1. COPPER REDUCTION METHOD C. NEOCUPROINE METHOD Cuprous ions + Neocuproine ↓ Cuprous-Neocuproine Complex (YELLOW or YELLOW-ORANGE) I. Chemical Methods 1. COPPER REDUCTION METHOD D. BENEDICT’S METHOD Modification of Folin Wu Used to detect and quantify reducing substances in body fluids Uses CITRATE and TARTRATE as stabilizing agent Negative result: BLUE Positive result: GREEN YELLOW BRICK RED PPT I. Chemical Methods 1. COPPER REDUCTION METHOD E. CLINITEST TABLET Modern version of Benedict’s method Uses URINE as sample Positive result: compare color reaction with a color chart Observe for PASS THROUGH PHENOMENON which is seen with increased glucose levels I. Chemical Methods 2. FERRIC REDUCTION METHOD a.k.a. HAGEDORN JENSEN METHOD Principle: INVERSE COLORIMETRY -Reduction of yellow ferricyanide to colorless ferrocyanide by reducing sugars Fe (CN6)-3 YELLOW ↓ Fe (CN6)-4 COLORLESS I. Chemical Methods 2. FERRIC REDUCTION METHOD Disappearance of color is measured at 400 nm Employed in AUTOANALYZERS I. Chemical Methods 3. CONDENSATION METHOD Principle: The aldehyde group of glucose condenses with aromatic amines in hot acetic acid solution to form colored derivatives A. ORTHO-TOLUIDINE METHOD a.k.a. DUBOWSKI METHOD MOST SPECIFIC NON-ENZYMATIC METHOD for glucose measurement MAJOR DISADVANTAGES: carcinogenic and teratogenic Glucose + Ortho-toluidine 100 °C ↓ Acetic acid Glycosylamine + Schiff’s base (BLUISH-GREEN measure at 620-630 nm) II. ENZYMATIC METHODS measures only GLUCOSE and not other reducing sugars 3 enzyme systems are commonly used to measure glucose: 1. Glucose Dehydrogenase 2. Glucose Oxidase A. Colorimetric B. Polarographic 3. Hexokinase Must know! Glucose exists either as alpha-glucose or beta-glucose. Alpha-glucose = 35% Beta-glucose = 65% Alpha glucose is converted into beta glucose using the enzyme mutarotase. II. Enzymatic Methods 1. GLUCOSE DEHYDROGENASE METHOD glucose is reduced to produce a chromophore that is measured spectrophotometrically or an electrical current amount of reduced nicotinamide adenine dinucleotide (NADH) generated is proportional to glucose concentration in the sample mutarotase II. Enzymatic Methods 2. GLUCOSE OXIDASE METHOD most specific enzyme reacting with only ß - D-glucose Glucose + O2 glucose ↓ oxidase Gluconic acid + H2O2 II. Enzymatic Methods 2. GLUCOSE OXIDASE METHOD A. Colorimetric Glucose Oxidase Method a.k.a. Saifer Gernstenfield Method Glucose + O2 glucose ↓ oxidase Gluconic acid + H2O2 H2O2 + chromogenic O2 receptor ↓ peroxidase Oxidized + H2O COLOR chromogen CHANGE II. Enzymatic Methods 2. GLUCOSE OXIDASE METHOD A. Colorimetric Glucose Oxidase Method The coupled reaction involved in glucose oxidase method is known as TRINDER’S REACTION High concentrations of uric acid, ascorbic acid, bilirubin, glutathione, creatinine, formalin, hemoglobin, tetracycline, l-cysteine, l-dopa, dopamine, methyldopa, and citric acid can cause FALSELY DECREASED results Presence of bleach and detergents, can cause FALSELY INCREASED results II. Enzymatic Methods 2. GLUCOSE OXIDASE METHOD B. Polarographic Glucose Oxidase Method Glucose + O2 glucose ↓ oxidase Gluconic acid + H2O2 H2O2 + chromogenic O2 receptor ↓ peroxidase Oxidized + H2O + O2 CLARK chromogen ELECTRODE II. Enzymatic Methods 2. GLUCOSE OXIDASE METHOD B. Polarographic Glucose Oxidase Method Oxygen depletion is measured and is proportional to the amount of glucose present. H2O2 is prevented from re-forming O2 by adding molybdate, iodide, catalase and ethanol Must know! Why glucose oxidase is NOT the reference method? Glucose oxidase is very specific, it only measures beta-glucose. Glucose oxidase is affected by reducing and oxidizing agents. II. Enzymatic Methods 3. HEXOKINASE METHOD REFERENCE METHOD/ GOLD STANDARD TEST MOST COMMONLY USED METHOD to determine serum glucose levels Measures BOTH alpha & beta D-glucose Glucose concentration is proportional to the rate of production of nicotinamide adenine dinucleotide phosphate (NADPH) II. Enzymatic Methods 3. HEXOKINASE METHOD Glucose + ATP --(hexokinase+Mg2+) glucose-6-phosphate + ADP Glucose-6-phosphate + NADP --(G6PD) 6-phosphogluconate + NADPH + H+ (340nm) *G6PD: glucose-6-phosphate-dehydrogenase enzyme II. Enzymatic Methods 3. HEXOKINASE METHOD ADVANTAGES: More accurate than the glucose oxidase method Not affected by ascorbic acid or uric acid May be performed on serum or plasma collected using heparin, EDTA, fluoride, oxalate, or citrate Other samples may be used: urine, CSF, and serous fluids II. Enzymatic Methods 3. HEXOKINASE METHOD DISADVANTAGES: Gross hemolysis and icterisia may cause a FALSE DECREASE in results. NONSPECIFIC- it reacts with any sugars with six carbon units (fructose, galactose, glucose) RBCs contain glucose-6-phosphate and intracellular enzymes that generate NADH causing POSITIVE INTERFERENCE. hemolyzed samples require a serum blank correction (subtraction of the reaction rate with hexokinase omitted from the reagent) LAB DETERMINATIONS FOR GLUCOSE 1.Random Blood Glucose (RBG) MONITORING TEST for blood glucose For determination of glucose at any time of the day Requested during: Insulin Shock Hyperglycemic Ketonic Coma EMERGENCY CASES ☺ 2.Fasting Blood Glucose (FBG) SCREENING TEST for serum glucose in the diagnosis of Diabetes Mellitus NPO (non-per-orem) for 8-10 hours Concept of RBG and FBG In a normal healthy individual, During a period of 8-10 hours of fasting, plasma glucose levels are within NORMAL Immediately after a meal, there is a fast INCREASE in the glucose level approx. 30 minutes after eating 1 hour after meal, glucose level reaches its PEAK. After 1 hour, glucose level starts to DECREASE and returns to NORMAL within 2 hours. LAB DETERMINATIONS FOR GLUCOSE 3. 2-Hour Post Prandial Blood Glucose Measures how well the body metabolizes glucose Based on the principle that glucose level in blood drawn 2 hours after a meal returns to normal in normal individuals while remains significantly increased in diabetic patients. 3. 2-Hour Post Prandial Blood Glucose Procedure: 1. Patient must eat a complete meal (75 grams of glucose) OR a solution containing 75 g of glucose is administered 2. Specimen for plasma glucose measurement is drawn 2 hours later Reference value must be 200 mg/dL and is confirmed on a subsequent day by either an increased random or fasting glucose level, the patient is diagnosed with diabetes LAB DETERMINATIONS FOR GLUCOSE 4. Glucose Tolerance Test (GTT) ▪ Multiple blood and urine glucose test ▪ Also referred to as CHALLENGE TEST ▪ Used to diagnose Gestational Diabetes Mellitus ▪ used to determine how well the body metabolizes glucose over a required period of time Gestational DM Arises when the extra-metabolic demands of the fetus put stress on the ability of the pregnant mother to produce adequate amounts of INSULIN for glucose utilization Glucose intolerance that develops during approximately 7% of all pregnancies Screening should be performed between 24-28 weeks of gestation LAB DETERMINATIONS FOR GLUCOSE 4. Glucose Tolerance Test (GTT) ▪ Based on the principle that, a normal individual when given a glucose challenge is capable of converting it to glycogen (for storage) and blood glucose returns to normal after 2 hours. ▪ Diabetic patients will remove glucose from the circulation at a slower rate due to insulin deficiency  Types of GTT A. ORAL GLUCOSE TOLERANCE TEST (OGTT) 1.JANNEY-ISAACSON METHOD (Single-Dose Method) MOST COMMON Procedure: 1. Fasting blood and urine samples are obtained 2. Glucose load is given 3. Blood and urine samples are collected for glucose measurements 30 minutes, 1 hour, 2 hours and 3 hours after  Types of GTT A. ORAL GLUCOSE TOLERANCE TEST (OGTT) 2. EXTON ROSE METHOD (Divided Oral Dose/Double Dose Method) Uses 100 grams of glucose load in 650 mL of water Procedure 1. Fasting blood and urine samples are obtained 2. First half of the total dose is taken 3. 30 minutes after the first load, samples are again taken 4. Second half of the dose is given 5. Samples are collected 30 minutes, 1 hour, 2 hours and 3 hours after Must know! In ORAL GLUCOSE TOLERANCE TEST (OGTT): The expected added plasma glucose after intake of glucose load are as follows: ▪ 30mins = 30-60 mg/dL above fasting ▪ 1-hour = 20-50 mg/dL above fasting ▪ 2-hour = 5-15 mg/dL above fasting ▪ 3-hour = fasting level or below Requirements for OGTT 1. Patient should be ambulatory (mobile) for 3 days before the test. CHO depletion and inactivity or bed rest impair glucose tolerance 2. The test should be performed after an overnight 8 to 10-hour fasting (not longer than 16 hours). Test is done only in the morning 3. Unrestricted diet of 150 g of CHO per day for 3 days prior to testing 4. Individual should not eat food, drink tea, coffee, or alcohol, or smoke cigarettes during the test, and should be seated. Requirements for OGTT 5. Glucose load 75 grams: for adults (WHO standard) 100 grams: for pregnant women (suspected GDM patient) 1.75g of glucose/kg body weight: for children Types of GTT B. INTRAVENOUS GLUCOSE TOLERANCE TEST (IVGTT) used for diabetics with gastrointestinal (GIT) disorders 0.5 g of glucose/kg body weight (given within 3 minutes) is administered INTRAVENOUSLY Collect blood after 5 minutes of IV glucose administration  Types of GTT B. INTRAVENOUS GLUCOSE TOLERANCE TEST (IVGTT) Indications for the use of IVGTT: ▪ Unable to tolerate large carbohydrate in the diet ▪ Presence of altered gastric physiology ▪ Previous operation or surgery of the gastrointestinal tract ▪ Presence of chronic malabsorption syndrome Reference Values for FBG CONVENTIONAL UNIT 70-100 mg/dL (serum) SI UNIT (International Standard) 3.85-5.50 mmol/L (serum) CONVERSION FACTOR Conventional to SI Unit: 0.055 Interpretation of Results (WHO) 1. FOR FASTING BLOOD GLUCOSE Non Diabetic: < 100mg/dL Impaired FBS: > 100mg/dl but < 126 mg/dL Diabetes Mellitus: > 126 mg/dL 2. FOR ORAL GLUCOSE TOLERANCE TEST Normal OGTT (2hr Glucose): < 140 mg/dL Impaired OGTT (2hr Glucose): 140-199 mg/dL Diabetes Mellitus (2hr Glucose): > 200 mg/dL Interpretation of Results (WHO) 3.DIAGNOSTIC CRITERIA FOR DIABETES MELLITUS RBG: > 200 mg/dL with symptoms of DM FBG: > 126 mg/dL 2-hr Post Prandial: > 200 mg/dL HbA1c : > 6.5% LAB DETERMINATIONS FOR GLUCOSE 5. GLYCOSYLATED HEMOGLOBIN (GLYCATED HEMOGLOBIN) (HbA1C) Hemoglobin A that is irreversibly glycosylated at one or both N- terminal valines of the beta chain Hemoglobin compound produced when glucose (a reducing sugar) reacts with the amino group of hemoglobin (a protein) Now the PREFERRED TEST FOR THE ASSESSMENT OF GLYCEMIC CONTROL It is a more reliable method on monitoring of LONG-TERM GLUCOSE CONTROL than RBG Used in monitoring how well patients maintain dietary and medication regimens Provides an index of average blood glucose levels over the past 2–3 months LAB DETERMINATIONS FOR GLUCOSE 5. GLYCOSYLATED HEMOGLOBIN (GLYCATED HEMOGLOBIN) (HbA1C) Sample of choice: non fasting WHOLE BLOOD drawn in EDTA tube Methods: Immunoassay Ion-exchange HPLC Electrophoresis Boronate affinity HPLC Enzyme methods Reference range: 4-6%  HbA1C Results > 6.5% on more than two occasions is indicative of Diabetes Mellitus For patients meeting treatment requirements: Measure HbA1C twice a year (every 6 months) For non-compliant patients: Measure HbA1C quarterly (every 3 months) NOTE: For every 1% change in HbA1C value, 35 mg/dL is added to plasma glucose level Factors Affecting HbA1C 1. Older RBC and iron deficiency anemia (IDA) results to HIGH HbA1c levels 2. Interference by carbamylated hemoglobin can occur with: Uremia Hypertriglyceridemia Hyperbilirubinemia 3. Interference by acetylated species: Salicylates administration Factors Affecting HbA1C 4. Factors which can affect accuracy of methodologies: Hemoglobinopathies (HbSS, HbSC, HbCC) Blood transfusions Chronic alcoholism Lead poisoning Opiate use IDA 5. Vitamin E causes falsely LOW results 4. Vitamin C causes falsely HIGH or LOW results Factors Affecting HbA1C Any condition associated with shortened red blood cell survival or lower mean red blood cell age will lower the HbA1c level as the result of reduced exposure to plasma glucose. Hemolysis Recovery from acute blood loss Transfusions Splenectomy * LAB DETERMINATIONS FOR GLUCOSE 6. FRUCTOSAMINE (GLYCATED ALBUMIN/ GLYCOSYLATED ALBUMIN) MOST WIDELY USED TO ASSESS SHORT-TERM GLYCEMIC CONTROL Used in monitoring glucose level at a shorter time interval (3-6 weeks) average half-life of the proteins is 2–3 weeks Advantages: serum samples and automated equipment may be used simple and low cost LAB DETERMINATIONS FOR GLUCOSE 6. FRUCTOSAMINE (GLYCATED ALBUMIN/ GLYCOSYLATED ALBUMIN) Useful for monitoring diabetic individuals with chronic hemolytic anemias and hemoglobin variants Should not be performed if the serum albumin level is low (≤3.0 mg/dL) Methods Use of strong acids (thiobarbituric acid) Nitrobluetetrazolium (NBT) Dye Test HPLC Point-of-Care Testing ▪ Use either glucose dehydrogenase (GDH) or glucose oxidase and are amperometric. ▪ Specimen: WHOLE BLOOD CAPILLARY GLUCOSE ▪ Uses a GLUCOMETER ▪ Hematocrit affects POCT glucose measurements. ▪ High hematocrit = lower glucose ▪ RBC glucose concentration is lower than plasma concentration ▪ NOT used to diagnose diabetes or hypoglycemic disorders ▪ For confirmation, laboratory measures of plasma glucose is required because of higher accuracy THANK YOU FOR LISTENING ☺