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Questions and Answers
What is the recommended time frame for obtaining fasting blood sugar after fasting?
What is the recommended time frame for obtaining fasting blood sugar after fasting?
Which specimen is the standard choice for glucose analysis?
Which specimen is the standard choice for glucose analysis?
What effect does sodium fluoride have when added to blood specimens?
What effect does sodium fluoride have when added to blood specimens?
At room temperature, how much does glucose metabolize per hour?
At room temperature, how much does glucose metabolize per hour?
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What is the principle behind the copper reduction method?
What is the principle behind the copper reduction method?
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Which fluid has a glucose concentration that is approximately 60-70% of plasma concentrations?
Which fluid has a glucose concentration that is approximately 60-70% of plasma concentrations?
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Which copper reduction method is known for its sensitivity and specificity?
Which copper reduction method is known for its sensitivity and specificity?
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What is the impact of bacterial contamination on glucose levels in a specimen?
What is the impact of bacterial contamination on glucose levels in a specimen?
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What color indicates a negative result in Benedict's method?
What color indicates a negative result in Benedict's method?
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How much lower are glucose levels in whole blood compared to serum or plasma?
How much lower are glucose levels in whole blood compared to serum or plasma?
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If processing of a specimen is delayed beyond 30 minutes, what is the recommended action?
If processing of a specimen is delayed beyond 30 minutes, what is the recommended action?
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Which method uses urine as a sample for detecting glucose levels?
Which method uses urine as a sample for detecting glucose levels?
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What is a common disadvantage of the Folin Wu Method?
What is a common disadvantage of the Folin Wu Method?
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In the Neocuproine method, what is formed when cuprous ions react with neocuproine?
In the Neocuproine method, what is formed when cuprous ions react with neocuproine?
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What is the main function of citrate and tartrate in Benedict's method?
What is the main function of citrate and tartrate in Benedict's method?
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Which of the following methods is a modification of the Folin Wu Method?
Which of the following methods is a modification of the Folin Wu Method?
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What is the primary principle behind the Hagedorn Jensen method?
What is the primary principle behind the Hagedorn Jensen method?
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Which wavelength is typically measured in the Ferric Reduction Method?
Which wavelength is typically measured in the Ferric Reduction Method?
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What is the major disadvantage of the Ortho-Toluidine Method?
What is the major disadvantage of the Ortho-Toluidine Method?
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Which enzyme is NOT part of the common enzyme systems for measuring glucose?
Which enzyme is NOT part of the common enzyme systems for measuring glucose?
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Which glucose form has the higher percentage in solution?
Which glucose form has the higher percentage in solution?
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What substance is produced when glucose is oxidized by the Glucose Oxidase method?
What substance is produced when glucose is oxidized by the Glucose Oxidase method?
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What does the Glucose Dehydrogenase Method use to determine glucose concentration?
What does the Glucose Dehydrogenase Method use to determine glucose concentration?
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Which chemical reaction in the Glucose Oxidase method indicates a color change?
Which chemical reaction in the Glucose Oxidase method indicates a color change?
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What type of specimen is used for point-of-care testing (POCT) glucose measurements?
What type of specimen is used for point-of-care testing (POCT) glucose measurements?
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How does high hematocrit affect point-of-care testing (POCT) glucose measurements?
How does high hematocrit affect point-of-care testing (POCT) glucose measurements?
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Which of the following statements is true regarding POCT glucose measurements?
Which of the following statements is true regarding POCT glucose measurements?
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What is the main limitation of using POCT glucose testing?
What is the main limitation of using POCT glucose testing?
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In comparison to plasma concentration, how does RBC glucose concentration typically rank?
In comparison to plasma concentration, how does RBC glucose concentration typically rank?
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Which factor must be considered when interpreting results from a glucometer?
Which factor must be considered when interpreting results from a glucometer?
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What must be performed for confirmation of glucose levels when using POCT?
What must be performed for confirmation of glucose levels when using POCT?
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Which of the following is NOT a reason POCT glucose is utilized in clinical settings?
Which of the following is NOT a reason POCT glucose is utilized in clinical settings?
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What type of sample is recommended for isotope dilution/mass spectrometry?
What type of sample is recommended for isotope dilution/mass spectrometry?
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Why is fasting typically not required before BUN analysis?
Why is fasting typically not required before BUN analysis?
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What is the conventional unit reference interval for BUN?
What is the conventional unit reference interval for BUN?
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What should be done if urine specimens cannot be analyzed within a few hours?
What should be done if urine specimens cannot be analyzed within a few hours?
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Which of the following is a suitable tube type for BUN determination?
Which of the following is a suitable tube type for BUN determination?
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Which sample is preferred for the measurement of glycated hemoglobin (HbA1C)?
Which sample is preferred for the measurement of glycated hemoglobin (HbA1C)?
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What is the reference range for HbA1C?
What is the reference range for HbA1C?
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How frequently should HbA1C be measured for patients meeting treatment requirements?
How frequently should HbA1C be measured for patients meeting treatment requirements?
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Which interference causes falsely high HbA1C results?
Which interference causes falsely high HbA1C results?
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What effect does a higher HbA1C level indicate regarding plasma glucose levels?
What effect does a higher HbA1C level indicate regarding plasma glucose levels?
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Which method is NOT commonly used for measuring HbA1C?
Which method is NOT commonly used for measuring HbA1C?
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Fructosamine is best used for monitoring which aspect of glycemic control?
Fructosamine is best used for monitoring which aspect of glycemic control?
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Which condition should NOT warrant the use of fructosamine testing?
Which condition should NOT warrant the use of fructosamine testing?
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Study Notes
Course Information
- Course title: MLS 301 - Clinical Chemistry 1
- Semester: First Semester
- Academic Year: 2024-2025
- Instructors: Zharina Leih Panopio-Atienza, Loren Deduyo
- Institution: College of Allied Medical Professions, Lyceum of the Philippines University - Batangas
Carbohydrates - Clinical Chemistry 1 Laboratory
- Topics include: glucose analysis methods, glycated hemoglobin, specimen selection, and error sources
- Specimens for carbohydrate analysis: whole blood, serous fluid, plasma, serum, urine, synovial fluid, CSF
- Standard clinical specimen: fasting venous plasma
Specimen Considerations (Glucose)
- Serum is suitable for glucose analysis if separation from cells occurs immediately after centrifugation
- Bacteria, white blood cells (WBCs), and red blood cells (RBCs) can consume glucose, potentially lowering results
- For delayed processing, use sodium fluoride (inhibits enzyme enolase involved in glycolysis)
- Refrigerated whole blood preservation: 2 mg of NaF per mL of whole blood prevents glycolysis for up to 48 hours
Specimen Considerations (General)
- Fasting blood glucose should be obtained after 8-10 hours of fasting (but not exceeding 16 hours)
- CSF glucose is approximately 60-70% 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 provides approximately 10-15% lower glucose levels than serum or plasma
- Venous blood glucose is 7 mg/dL lower than capillary blood glucose due to tissue metabolism
- Capillary blood glucose is equivalent to arterial blood glucose
- Glucose metabolism at room temperature: 7 mg/dL/hour
- Glucose metabolism at 4°C: 2 mg/dL/hour
- Bacterial contamination or leukocytosis increase glucose metabolism rate
- In serum samples without bacterial contamination or leukocytosis, results are clinically acceptable up to 90 minutes prior to serum separation from cells
Glucose Methodologies
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Chemical methods (oxidation-reduction):
- Copper reduction (oldest method) - Folin Wu, Nelson Somogyi, Neocuproine, Benedict's, Clinitest
- Ferric reduction (Hagedorn-Jensen)
- Condensation method - Ortholutidine(Dubowski)
- Enzymatic methods: Glucosidase, Hexokinase-G6PD
Glucose Methodologies - Details
-
Copper Reduction
- Glucose and other reducing sugars convert cupric to cuprous ions in the presence of heat and alkali, causing color formation.
- CuSO4 + Glucose + Heat + Alkali → Cu2O + Oxidized substance
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Folin Wu Method
- Sensitive, but not specific. Non-glucose reducing substances also react with the test.
- Cuprous ions + Phosphomolybdate → Phosphomolybdenum BLUE
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Nelson Somogyi Method
- Sensitive and specific. Non-glucose reducing substances are adsorbed using barium sulfate.
- Cuprous ions + Arsenomolybdate → Arsenomolybdenum BLUE
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Neocuproine Method
- Cuprous ions + Neocuproine → Cuprous-Neocuproine complex(yellow or yellow-orange)
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Benedict's Method
- Modification of Folin Wu. Detects and quantifies reducing substances in body fluids using citrate and tartrate as stabilizing agents.
- Negative: BLUE; Positive: GREEN-YELLOW/BRICK RED PPT
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Clinitest Tablet
- Modern version of Benedict's method, using urine as the sample. Positive result is indicated by comparing the color reaction with a color chart.
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Hagedorn-Jensen Method
- Principle is inverse colorimetry
- Reduction of yellow ferricyanide to colorless ferrocyanide by reducing sugars
- Fe(CN)6-3 (YELLOW) → Fe(CN)6-4 (COLORLESS)
- Color disappearance is measured at 400nm.
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Dubowski (Ortho-toluidine) Method
- Most specific non-enzymatic method for glucose measurement
- Glucose + Ortho-toluidine + Acetic Acid → Glycosylamine + Schiff's base (bluish-green product at 620-630nm)
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Enzymatic Methods
- Glucose Dehydrogenase Method- Amount of reduced nicotinamide adenine dinucleotide (NADH) generated is proportional to glucose concentration in the sample.
- Glucose Oxidase Method- Most specific enzyme reacting with only B-D-glucose. Glucose + O2 → Gluconic acid + H2O2
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Colorimetric Glucose Oxidase Method (Saifer Gernstenfield Method) Glucose + O2 → Gluconic acid + H₂O2 H₂O₂ + chromogenic O₂ receptor → Oxidized chromogen + H₂O + O2
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Polarographic Glucose Oxidase Method Glucose + O2 → Gluconic acid + H₂O2 H₂O₂ + chromogenic O₂ receptor → Oxidized chromogen + H₂O + O2
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Hexokinase Method - Reference method/gold standard test for serum glucose levels. Measures both alpha and beta D-glucose. Glucose concentration is proportional to the rate of production of nicotinamide adenine dinucleotide phosphate (NADPH)
Other Lab Determinations for Glucose
- Random Blood Glucose (RBG): Monitoring test for daily glucose levels
- Fasting Blood Glucose (FBG): Screening test for serum glucose to diagnose diabetes mellitus; non-per-oral intake for 8-10 hours
- 2-Hour Postprandial Blood Glucose: Measures how well the body metabolizes glucose after a meal; 75 grams of glucose intake, measurement 2 hours later
- Glucose Tolerance Test (GTT): Multiple blood and urine glucose tests to diagnose gestational diabetes mellitus; used to analyze how well the body handles glucose over a period.
- Types of GTT (OGTT): Janney-Isaacson Method (single dose method -most common), Exton Rose Method (divided oral-dose/double dose method)
Glycosylated Hemoglobin (HbA1c)
- Hemoglobin A that is irreversibly glycosylated at one or both N-terminal valines of the beta chain
- Formed when glucose reacts with the amino group of hemoglobin (a protein)
- Preferred test for long-term glycemic control
- More reliable than RBG for monitoring long-term glucose control
- Measures average blood glucose levels over the past 2-3 months
Fructosamine (Glycated Albumin/Glycosylated Albumin)
- Most widely used to assess short-term glycemic control
- Monitoring glucose level at a shorter time interval, typically 3-6 weeks
- Serum samples & automated equipment - simple & cost-effective -Useful for diabetics with chronic hemolytic anemia, hemoglobin variants
- Should not be performed on serum albumin levels less than 3.0 mg/dL
Point-of-Care Testing
- Uses either glucose dehydrogenase (GDH) or glucose oxidase, amperometric
- Specimen: whole blood capillary glucose; uses a glucometer
- Hematocrit affects POCT glucose measurements
- High hematocrit = lower glucose
- Not used to diagnose diabetes or hypoglycemic disorders. Laboratory measures of plasma glucose are required for higher accuracy
Non-Protein Nitrogenous Substances
- Key topics: urea, uric acid, creatinine, creatinine, and ammonia
- Specimen requirements: collection, transport, and storage for urea, uric acid, creatine and ammonia.
- Commonly used methods for the determination of urea, uric acid, creatinine.
- Sources of error and variability in these methods & effects of these on clinical utility
- Reference intervals for urea, uric acid, creatinine & ammonia in plasma & urine.
- Effects of age and gender on these values.
Urea
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Lab Methodologies & Analytical Techniques:
- Direct Methods: directly measure urea
- Indirect Methods: measure nitrogen content of urea (Blood Urea Nitrogen).
- Microkjeldahl (indirect): used as a classical reference method for urea; digests Nitrogen to NH3 using a specific acid mixture
- Diaceyl Monoxime (DAM): employed in autoanalyzers for direct urea measurement
- Enzymatic (indirect): use enzyme urease (coupled with Nessler or Berthelot reactions) to determine urea indirectly by measuring ammonia produced
- Isotope Dilution/ Mass Spectrometry (IDMS): gold standard, but expensive
Creatinine
-
Lab Methodologies & Analytical Techniques:
- Jaffe Reaction: Direct method, creatinine in filtrate is reacted with alkaline picrate to form a coloured complex
- Reagent: Alkaline picrate (picric acid dissolved in 10% NaOH) needs to be freshly prepared
- Disadvantages: not specific to creatinine; interferences
- Removal of Interferences: Lloyd's reagent (sodium aluminum silicate) or Fuller's Earth reagent eliminate interferences
- Kinetic Jaffe Method: Measure the rate of color development to eliminate interferences from non-creatinine substances
- Enzymatic Methods: (creatinine amidinohydrolase or creatinine iminohydrolase) methods for creatinine measurements
Uric Acid
-
Lab Methodologies & Analytical Techniques:
- Cyanide Method (REDOX): utilizes Folin, Brown, Newton, and Benedicts methods; uric acid + PTA + NaCN → tungsten blue
- Sodium Carbonate Method: Archibald, Henry, and Caraway methods; uric acid + PTA + Na2CO3 → tungsten blue
- Enzymatic Method (Uricase): simplest and most specific method; uric acid → allantoin using the uricase enzyme
Ammonia
- Lab Methodologies & Analytical Techniques:
-
Two-step Approach: ammonia is isolated from the sample and then measured
- One-step approach: ammonia is measured directly from the sample using enzymatic methods or ion-selective electrodes
- Enzymatic Method (Glutamate Dehydrogenase method): uses a-ketoglutarate, ammonia, and NADPH to measure ammonia indirectly
Lipid Analysis
- Forms of Lipid: Triglycerides, Cholesterol, Phospholipids, Fatty Acids, Fat-Soluble Vitamins
- Methodologies & Specimen Considerations:
- Lipids: Fasting for at least 12 hrs recommended for most testing -Measurements frequently performed on non-fasting individuals using a serum separator tube -Avoid alcohol intake 2 days before tests -Various methods of analysis exist, some requiring specialized equipment
- Posture: Posture changes alter TAG, therefore subjects should be seated for 5 minutes prior -Avoid prolonged tourniquet use to prevent hemoconcentration
Cholesterol
- Methodologies & Specimen Considerations:
- Methodologies: various chemical (Liebermann-Burchard, Salkowski, Colorimetric) and enzymatic (Cholesterol oxidase) methods available.
- Specimen Considerations: Fasting for at least 12 hours -Refrigerated samples if analysis is delayed for several days
- Avoid hemolyzed and icteric samples
- Methods: chemical methods involve dehydration and oxidation of cholesterol to form a colored compound
- One-Step method: direct colorimetric methods, e.g. Pearson, Stern, & Mac Gavack Methods
Lipoproteins
- Classifications: Density, Rate of Migration, and Content
- Density: High-density lipoproteins (HDL), Low-density lipoproteins(LDL), Very-low-density lipoproteins (VLDL), Chylomicrons
- Rate of Migration: HDL → alpha, VLDL → pre-beta, LDL → beta, Chylomicrons → gamma
- Content: Chylomicrons (Triglycerides, Gammaglobulin), VLDL (Triglycerides, Pre-Beta-globulin), LDL (Cholesterol, Beta-globulin), & HDL (Cholesterol, Alpha-globulin)
Lab Methodologies (Lipoproteins and related items)
-
Electrophorosis: Used to differentiate lipoproteins based on charge and mass in an alkaline medium using agarose gel -Lipid stains, including Oil Red O, Fat Red 7B, Sudan Black B, and Scharlach R, are used for visualization
-
Ultracentrifugation: Reference method for lipoprotein quantification; employs density gradients to separate lipoproteins based on their density, and then VLDL, LDL, HDL are quantifiable based on the analysis of resulting fractions: - Reagents: potassium bromide solution (density = 1.063) are used
- Frozen samples are discouraged
-
CDC Reference (Abell-Kendal) method for HDL: a 3 step procedure first ultra-centrifuging to precipitate VLDL and then precipitate LDL and measure cholesterol in remaining supernatant with the Abell-Kendal method.
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Various methods for LDL-c calculation (Friedewald and DeLong): Methods used to estimate LDL-c based on measured values of TC, HDL-c, and triglycerides -VLDL is estimated, if applicable
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Standing Plasma Test: A method for qualitatively determining cholesterol and TAG levels based on observations of the characteristics of the supernatant (presence/absence of layers, clarity, homogeneity, colour)
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NCEP Guidelines for HDL Cholesterol and LDL Cholesterol reference ranges (in mg/dL)
Additional Notes
- Women have higher HDL-C while men have higher LDL and triglycerides on average, which is partly due to estrogen.
- Both genders show increased total cholesterol, LDL-C, and triglycerides concentrations with age.
- The factors that contribute to coronary heart disease, as noted by the NCEP, include age, premature menopause (for females), smoking, family history of premature CHD, high LDL-c, low HDL-C, diabetes mellitus, and metabolic syndrome
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