EQ 22

• Cholesterol


  Accounts for most of the sterol in plasma, consists of both free (unesterified, 30-40%) and esterified (60-70%) forms
• Cholesterol and triglycerides


  • The most commonly ordered lipid tests in the laboratory, used in the diagnosis and management of lipoprotein disorders as well as in cardiovascular disease
• Cholesterol determination


  1. Cholesterol esters converted to free cholesterol and fatty acids by cholesterol esterase

  2. Cholesterol oxidized to cholest-4-en-3-one and hydrogen peroxide by cholesterol oxidase

  3. Hydrogen peroxide reacts with 4-aminoantipyrine and phenol, catalyzed by peroxidase, to form a red quinoneimine dye
• The intensity of the red color produced is directly proportional to the total cholesterol in the sample when read at 500 nm
• Materials and equipment


  • Venipuncture set
  • Test tubes
  • Cuvettes
  • Pipettes
  • Test tube rack
  • Labeling tape
  • Nescofilm/parafilm
  • Applicator sticks
  • Timer
  • Centrifuge
  • Water bath
  • Spectrophotometer
• Reagent composition


  • Cholesterol esterase 150U/L
  • Cholesterol oxidase 150 U/L
  • Peroxidase 2500 U/L
  • 4-aminoantipyrine 0.3mM
  • Phenol 15mM
  • Phosphate buffer pH 6.8
  • Non reactive stabilizers and preservatives
• Cholesterol standard


  200 mg/dL
• The reagent is ready to use, stored at 2-8 C, and stable until the expiration date
• Do not use if the reagent is turbid, or when the reagent does not meet stated performance parameters
• Reagent contains sodium azide, a poison that may react with lead and copper plumbing to form highly explosive metal azides
• Specimen


  • Use nonhemolyzed serum
  • Cholesterol in serum is stable for seven days at room temperature and six months when frozen and protected against evaporation
  • Improper techniques in blood collection affect the cholesterol measurement
  • A number of drugs and substances affect concentrations of cholesterol
• Assay conditions


  • Wavelength: 500 - 520 nm
  • Optical path: 1 cm
  • Temperature: 37 C
  • Measurement against reagent blank
• Procedure


  1. Label test tubes

  2. Pipette cholesterol reagent into each tube and prewarm at 37 C for at least 5 minutes

  3. Add sample to respective tubes

  4. Cover and mix by gentle swirling

  5. Incubate the tubes at 37 C water bath for 5 minutes

  6. Zero the spectrophotometer with Blank at 500 nm

  7. Read and record absorbance of all tubes
• If the spectrophotometer requires a final volume greater than 1.0 mL, use 0.025 mL (25 uL) of sample to 3.0 mL of reagent
• Grossly lipemic serum requires a "sample blank" to obtain the correct reading
• Linearity


  The reagent is linear up to 700 mg/dL total cholesterol
• Calibration


  1. Aqueous standards or an appropriate serum calibrator can be used

  2. The procedure should be calibrated according to the instrument manufacturer's instructions

  3. If control results are out of range, the procedure should be recalibrated
• Quality Control


  • Known high and low values of cholesterol controls should be included in each set of assays
  • Failure to obtain the proper range of values in the assay of control material may indicate reagent deterioration, instrument malfunction or procedural errors
• Performance Characteristics


  • Linearity: 700 mg/dL
  • Sensitivity: an absorbance change of 0.001 at 500 nm corresponds to 0.5 mg/dL
  • Specificity: Cholesterol Oxidase is not totally specific for cholesterol, but other analogs do not normally occur in appreciable amounts in serum
• Calculations


  1. Concentration of test (mg/dL) = Absorbance of test / Absorbance of standard x Concentration of standard

  2. To obtain results in SI units (mmol/L), multiply the results in mg/dL by 0.02586
• Expected Values


  • Desirable cholesterol: <200 mg/dL
  • Borderline-high cholesterol: 200 - 239 mg/dL
  • High cholesterol: >240 mg/dL
• Principle of Lipid Phase control

  1. Cholesterol ester -> Cholesterol esterase -> free cholesterol + fatty acids
  2. Cholesterol + O2 -> Cholesterol oxidase -> cholest-4-en-3-one + H2O2
  3. 2H2O2 + -Aminoantipyrine + phenol -> Peroxidase -> quinoneimine + 4 H2O
• What is the colored of the end product of the assay?

  Red due to phenol
• Specimen consideration

  Use of anticoagulant such as EDTA is recommended
• Citrate and Fluoride


  Alters osmolality
• Heparin


  interferes with electrophoresis
• Specimen Collection

  • Fast about atleast 12 hours
  • Can consume water but not food
  • Atleast inhibit Ethanol consumption atleast 48 hours
• Specimen Collection

  • Fast about atleast 12 hours
  • Can consume water but not food
  • Atleast inhibit Ethanol consumption atleast 48 hours
• Ethanol consumption

  If ingested causes rise in triglyceride levels
• Reject specimen if

  1. Hemolyzed at >0.05
  2. Icteric sample
  3. Presence of bilirubin at levels of 5 mg/dL
• Acceptable CV for LIpids

  1. Total Cholest: 3%
  2. LDL Cholest: 4%
  3. HDL Cholest: 4%
  4. Triglycerides: 5%
• Avoid what?

  Water contamination, especially for cholesterol assays: Liebermann-Burchard
• Cholesteryl esters are hydrolyzed with alcoholic potassium hydroxide (KOH), unesterified cholesterol is extracted with petroleum ether and measured with the Liebermann-Burchard reagent using purified cholesterol standards.This method can be accurate within about 0.5% of true value.
• Modification of the Abell-Kendall Method


  reference method for cholesterol used by the CDC
• Storage

  • Short term storage (1-2 mtnhs) = 20 degrees
  • Long term storage -70 degrees
• Where do you get your samples?

  Lipids in capillary is a little lower than venous

  Venous blood is recommended
• Posture

  When a standing patient reclines, extravascular water transfers to the vascular system and dilutes nondiffusable plasma constitutents

  • Decreases plasma as much as 10% in the concen of HDL-C,LDL-C,T
• Posture

  Recommends patients to be seated atlest 5 mins prior sampling to prevent hemoconcentration
• Biological variations

  1. Cholesterol levels rise with age starting from early adulthood in both sexes
  2. Before ascertaining a patients TC level, s/he should be on the usual diet for 2 weeks
  3. Dietary intake of saturated fat and cholesterol significantly influences plasma lipid levels
  4. slightly higher in the winter (Robinson, 1992) - seasonal variation
  5. Women have lower levels than men, except in childhood and after the early fifties