MLS 301 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  COPYRIGHT NOTICE This materi...

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