Untitled Quiz

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

• 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

• Folin Wu Method

  • Sensitive, but not specific. Non-glucose reducing substances also react with the test.
  • Cuprous ions + Phosphomolybdate → Phosphomolybdenum BLUE

• Nelson Somogyi Method

  • Sensitive and specific. Non-glucose reducing substances are adsorbed using barium sulfate.
  • Cuprous ions + Arsenomolybdate → Arsenomolybdenum BLUE

• Neocuproine Method

  • Cuprous ions + Neocuproine → Cuprous-Neocuproine complex(yellow or yellow-orange)

• 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

• 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.

• 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.

• 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)

• 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

• Colorimetric Glucose Oxidase Method (Saifer Gernstenfield Method) Glucose + O2 → Gluconic acid + H₂O2 H₂O₂ + chromogenic O₂ receptor → Oxidized chromogen + H₂O + O2

• Polarographic Glucose Oxidase Method Glucose + O2 → Gluconic acid + H₂O2 H₂O₂ + chromogenic O₂ receptor → Oxidized chromogen + H₂O + O2

• 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

• 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.

• 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

• 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)

• 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