CLINICAL LAB SCIENEC

CHAPTER 14: CLINICAL CHEMISTRY 377
Total Cholesterol and HDL Procedure
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
Reagent blank
Total cholesterol standard (1 mL reagent and 10 μL standard—200 mg/dL)
Total cholesterol unknown (1 mL reagent and 10 μL serum)
HDL cholesterol standard (1 mL reagent and 25 μL standard—50 mg/dL)
HDL cholesterol unknown (1 mL total cholesterol reagent and 25 μL supernatant
from the serum preparation precipitate)
6. Mix well the contents of tubes 1 – 5 by using a vortex mixer if available, or mix
by swirling gently or by gently tapping the bottoms of the tubes. Note that volumes
may be increased if the instrument requires volumes greater than 1.0 mL,
which would require larger volumes of reagent and sample in tubes 1 – 5.
Total Cholesterol
Reagent Blank (RB) Standard (S) Sample (U)
Reagent 1.0 1.0 1.0
Standard – 0.01 –
Sample – – 0.01
HDL Cholesterol
Reagent Blank (RB) Standard (S) Sample (U) from the
precipitate
Reagent 1.0 1.0 1.0
Standard – 0.025 –
Sample – – 0.025
7. Allow all tubes to stand for 10 minutes at room temperature.
8. Pipette 1.0 mL color reagent into each tube and again mix by gently tapping the
bottom of the tube. Incubate the tubes at room temperature for 10 minutes or in a
water bath or heat block set at 37˚C (optional). The reaction requires 10 minutes
at room temperature, or approximately 3 minutes at 37˚C.
9. Pour into cuvettes for measuring absorbances (if the spectrophotometer is
equipped with a flow-through cuvette, aspirate the contents of the test tube
directly).
10. Within 60 minutes, read and record the absorbance values for the standard (S)
and the unknowns (U) versus the reagent blank (RB) at 500 nm.
11. Calculate the values for controls and the patient specimens (unknowns) using the
following equation.
Total Cholesterol Calculation (Manual Method)
Total Cholesterol (mg/dL) =
Abs of unknown (Au) × 200 (concentration of
the standard) (mg/dL)
Abs of standard (As)
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