MLS 301 - Clinical Chemistry 1 Lecture Notes 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 ☺ MLS 301 – CLINICAL CHEMISTRY 1 First Semester AY 2024-2025 Zharina Leih Panopio-Atienza | Loren Deduyo Instructors College of Allied Medical Professions  COPYRIGHT NOTICE This material has been reproduced and communicated to you or on behalf of Lyceum of the Philippines University- Batangas pursuant to PART IV: The Law of Copyright of Republic Act RA 8293 or the "Intellectual Property Code of the Philippines". The University does not authorize you to reproduce or communicate this material. The material may contain works that are subject to copyright protection under RA 8293. Any reproduction, and/or communication of the material by you may be subject to take legal action against such infringement. Do not remove this notice. This material is prepared by the Faculty of the Department of Medical Laboratory Science, LPU-Batangas College of Allied Medical Professions, solely for the use of students enrolled in MLS 301- Clinical Chemistry 1 for the A.Y. 2024- 2025. Please do not distribute without permission.  NON-PROTEIN NITROGENOUS SUBSTANCES College of Allied Medical Professions Medical Laboratory Science  OBJECTIVES State the specimen collection, transport, and storage requirements necessary for determinations of urea, uric acid, creatinine, creatine, and ammonia. Discuss commonly used methods for the determination of urea, uric acid, creatinine, creatine, and ammonia in plasma and urine. Identify sources of error and variability in these methods and describe the effects on the clinical utility of the laboratory measurements. Recognize the reference intervals for urea, uric acid, creatinine, and ammonia in plasma and urine. State the effects of age and gender on these values.  UREA Lab Methodologies & Analytical Techniques Laboratory Methodologies & Analytical Techniques A. Direct Methods- directly measure urea B. Indirect Methods- measure the nitrogen content of Urea (Blood Urea Nitrogen) CO (NH2)2 C - 1 x 12 = 12 60g/mole = 28 g Nitrogen O -1 x 16 = 16 60/28 = 2.14 N - 2 x 14 = 28 H–4x1 = 4 BUN x 2.14 = UREA 60g/mole 1. Micro - Kjeldahl (Indirect) § CLASSICAL REFERENCE METHOD FOR UREA a. Digest Nitrogen to NH3 (Kjeldahl Process) § Uses an acid digestion mixture § Nitrogen ammonia (NH3) § Principle: Kjeldahl- Nitrogen in PFF is converted to ammonia using hot concentrated sulfuric acid in the presence of a catalyst. b. Measurement of ammonia 1. Micro - Kjeldahl (Indirect) § Measurement of ammonia 1. Micro-Kjeldahl Nessler Ø Nessler’s Reaction (Nesslerization) § NH3 + K2Hg2I2 gum ghatti NH2Hg2I2 § dimercuric potassium iodide (Nessler’s reagent) 2. Micro-Kjeldahl Berthelot Ø Berthelot Reaction § NH3 + phenol + hypochlorite indophenol blue BUN result x 2.14 = UREA  2. DAM: Diacetyl Monoxime (Direct) § EMPLOYED IN AUTOANALYZERS § UREA + DAM strong acid yellow diazine derivative § Reaction is intensified by: § ferric ions and thiosemicarbazide à intense red color formed is measured at 540 nm § Utilizes the FEARON Reaction ADVANTAGES: § Simple § Direct measurement of UREA § Shows no interference by ammonia Laboratory Methodologies & Analytical Techniques § Color reaction of NH3 – Berthelot Reaction § indophenol blue § Color reaction of N – Nessler’s Reaction § Low to moderate: yellow § High: orange brown § Color reaction of UREA- Fearon Reaction § yellow 3. Enzymatic Method (Indirect) a. Urease-Nessler Urea Urease CO2 + NH3 + K2Hg2I2 (yellow) BUN result x 2.14 = UREA 3. Enzymatic Method (Indirect) b. Urease-Berthelot Urea Urease CO2 + NH3 + phenol hypochlorite à indophenol blue (630nm) BUN result x 2.14 = UREA 3. Enzymatic Method (Indirect) c. Coupled Urease/Glutamate Dehydrogenase (GLDH) Method § CANDIDATE REFERENCE METHOD 4. ISOTOPE DILUTION/ MASS SPECTROMETRY -GOLD STANDARD -used only as a REFERENCE METHOD due to its high cost  PRECAUTIONS & SPECIMEN CONSIDERATIONS § Specimens: Plasma, Serum or Urine § NONHEMOLYZED sample is recommended § Highly affected by protein diet § BUT the effect of a recent protein meal is minimal § fasting sample is NOT required usually § Gray and blue-top tubes are not suitable for BUN determination § Fluoride and citrate are inhibitors of UREASE enzyme  PRECAUTIONS & SPECIMEN CONSIDERATIONS § Avoid contamination with ammonium salts à can become ammonia § Avoid prolonged standing of sample àbecause urea will be converted to ammonia § Urea is susceptible to bacterial decomposition § Specimens (particularly urine and timed urine samples) that cannot be analyzed within a few hours should be refrigerated.  Reference Interval / Range § BUN: § Conventional unit: § 8 – 23 mg/dL § SI unit § 2.9 – 8.2 mmol/L § Conversion Factor: § Conventional to SI unit à 0.357 § Remember! ü Values are lower in children ü Males have slightly higher values compared to females  CREATININE Lab Methodologies & Analytical Techniques Laboratory Methodologies & Analytical Techniques 1. Direct JAFFE Reaction Method (1886) Creatinine in protein free-filtrate is reacted with alkaline picrate to form a colored complex Creatinine (serum) alkaline↓picrate red-orange tautomer of creatinine picrate § Reagent: ALKALINE PICRATE §picric acid (trinitrophenol) dissolved in 10% NaOH §should be freshly prepared because prolonged standing will convert picric acid to picramic acid DISADVANTAGES: Falsely Falsely § Not specific for Increased Decreased Ketones Bilirubin creatinine Hemoglobin Glucose § Interferences à Fructose Protein Urea Ascorbic acid Cephalosporins § To remove interferences use: § (Method of Hare) LLOYD’S reagent - sodium aluminum silicate § FULLER’S EARTH reagent - aluminum magnesium silicate § These two reagents eliminate the interferences making the method both specific and sensitive. Laboratory Methodologies & Analytical Techniques 2. Kinetic Jaffe Method § Differential rate of color development of non-creatinine chromogens thus eliminating interferences § Popular, inexpensive, rapid and easy to perform § Requires automated equipment § PFF is not needed, serum is used directly Laboratory Methodologies & Analytical Techniques 3. ENZYMATIC methods § No interference from glucose and other Jaffe chromogens a. Creatinine Iminohydrolase Method Creatinine Creatinine iminohydrolase NH4 + N-methylhydantoin NH4 + 2-oxoglutarate + NADH Glutamate dehydrogenase à glutamate and NAD ***Decrease in absorbance at 340nm reflects the concentration of creatinine 3. ENZYMATIC methods b. Creatinine Amidohydrolase Method Creatinine Creatinine amidohydrolase Creatine Creatine imidinohydrolase and sarcosine oxidase H202 2,4-dichlorophenolsulfonate + H202 horseradish↓ peroxidase colorless polymer (510 nm) 3. ENZYMATIC methods Other coupled enzymatic methods adapted for employed on a dry slide analyzer uses: -Creatininase (creatinine amidohydrolase) -Creatinase (creatine amidinohydrolase) -Sarcosine oxidase -Peroxidase Laboratory Methodologies & Analytical Techniques 4. ISOTOPE DILUTION/MASS SPECTROMETRY (IDMS) ULTIMATE REFERENCE STANDARD for creatinine measurement  PRECAUTIONS & SPECIMEN CONSIDERATIONS § Specimens: Plasma, Serum, or Urine § Hemolyzed samples should be avoided § considerable amounts of non-creatinine chromogens are present in RBC’s. § Lipemic and icteric samples produce erroneous results. § If stored at proper conditions it must be maintained at a pH of 7.0  PRECAUTIONS & SPECIMEN CONSIDERATIONS § Fasting sample is NOT required, although high-protein ingestion may transiently elevate serum concentrations. § Urine should be refrigerated after collection § Urine should be frozen if longer storage than 4 days is required. § Effects of some drugs: § Dopamine interferes with Jaffe and enzymatic methods § Lidocaine causes a positive bias in some enzymatic methods  Reference Interval / Range § Serum or plasma CREATININE § Adult: 0.6–1.2 mg/dL (53–106 μmol/L) § Children