CLINICAL LAB SCIENEC

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ESSENTIALS OF CLINICAL LABORATORY SCIENCE
In the Lab
6. Spectrophotometer capable of absorbance readings at 500 nm
7. Stanbio kit for enzymatic determination of total cholesterol. Total cholesterol
standard (200 mg/dL) and HDL standard (50 mg/dL) and HDL precipitating reagent
are included in the Stanbio kit.
8. Water bath or heat block set at 37°C (optional) (The reaction takes 10 minutes at
room temperature, and approximately 3 minutes at 37°C.)
9. Cuvettes
10. 13 × 100 test tubes and test tube rack
11. Waterproof sharp marker for labeling test tubes
12. Vortex mixer
13. Electronic timing device or clock/watch (exact time is not critical)
14. 2 or 3 levels of control samples to insure precision (recommended)
Procedure
1. Wash hands and don gloves.
2. Assemble necessary equipment and supplies.
3. Assemble the micropipetter with tips for accurate pipetting of the standards, controls,
and patient specimens. The tips should be changed between each sample
(including standards and controls) since carryover is possible between specimens.
Accurate pipetting is essential since extremely small amounts of standard,
controls, and patient specimens are required for this procedure.
4. Label the test tubes as follows for proper identification of blanks, standards, controls,
and unknowns:the first tube “RB” for the reagent blank; the second tube
“TCS” for the total cholesterol standard; the third tube “TCU” for the total cholesterol
unknown sample along with the reagent; the fourth tube “HDLS” for the HDL
standard; and the fifth tube “HDLU” for the HDL unknown sample taken from the
precipitate along with the reagent.
a. This procedure will require 5 tubes when only one unknown is being tested.
b. The only difference between HDL determination and total cholesterol is the
amount of sample and the standard.
c. All of the tests may be read using the same wavelength of 500 nm, with only
one blank.
5. Performing Precipitate for HDL Cholesterol
a. This is accomplished by adding an amount of serum, in accordance with the
test kit manufacturer’s instruction, to a precipitating reagent. This mixture is
centrifuged, and after centrifugation the supernatant from which the low density
lipoproteins (LDL) have been removed (precipitated out of the serum) is
used as the patient sample. If only total cholesterol or only HDL is being performed,
use the tables following the one entitled “Total Cholesterol and HDL
Procedure.”
b. A vortex mixer is preferable, particularly for mixing the serum for an HDL with
the precipitating reagent.
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