CLINICAL LAB SCIENEC

CHAPTER 14: CLINICAL CHEMISTRY 371
3. Assemble the pipetter with tips for accurate pipetting of the standards, controls,
and patient specimens.
a. The tips should be changed between each sample (including standards and
controls) since carryover is possible between specimens.
b. 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: Label one tube “RB” for the blank specimen and one tube
“S” for the standard sample, and one or more tubes “C (number)” for the control
samples (e.g., C-1 and C-2 to differentiate between normal and abnormal controls).
The unknown samples [Sample (U) in the chart below] should be labeled by
patient name or other identifier such as a patient number.
5. Pipette the following volumes into the 13 × 100 tubes and mix well. Note that
the reagent is measured in mL and the specimens, standards, and controls are
measured in μLs (1000 μLs comprise 1 mL). The volumes indicated below may be
increased if the spectrophotometer requires a volume greater than 1.0 mL.
Reagent Blank (RB) Standard (S) Sample (U) Control (C)
Reagent 1.0 1.0 1.0
Standard – 0.01 –
Sample – – 0.01
6. Use the vortex mixer or gently swirl the mixture to briefly mix the contents of
each tube. Take care not to lose any of the sample and reagent mixture.
7. Allow all tubes to stand for 10 minutes.
8. Arrange the cuvettes in the order in which they will be read after filling them from
the test tubes.
9. Insert the reagent blank into the well of the spectrophotometer and adjust the
absorbance to “0.”
10. Within 60 minutes of starting the procedure, read and record the absorbance values
for the standard (S), control (C), and the unknowns (U) versus the reagent blank (RB)
at 550 nm.
11. Calculate the values for the controls and the patient specimens (unknowns) by
the following equation.
Protein Calculation (Manual Method)
Protein (g/dL) = Abs of unknown (Au) × 10 (concentration of the
standard) (mg/dL)
Abs of standard (As)
12. Discard all supplies used for the procedure in the appropriate containers.
A disinfectant should be used to clean the work surfaces and equipment should
be cleaned and restored to its former position. Gloves should be discarded
appropriately and the hands washed thoroughly in accordance with established
policies.
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