CLINICAL LAB SCIENEC

CHAPTER 13: HEMATOLOGY AND COAGULATION 347
3. Label the test tubes as follows for proper identification of blanks, standards, controls,
and unknowns: Label one tube “RB” for the reagent blank specimen, 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 should be labeled by patient name, patient number,
or other identifier.
4. Pipette 5.0 mL cyanmethemoglobin reagent into the tubes labeled “RB” and
“S” and into each sample tube.
5. Add by pipette 0.02 mL of the standard specimen to the tube labeled “S.” Mix well.
6. Add by pipette 0.02 mL of the controls and unknown specimen to the sample
tubes. Mix well.
7. Using a micropipetter, add 0.02 mL of deionized water (in place of a specimen) to
the tube labeled “RB.” Mix well.
8. Allow the specimens to stand at room temperature (15°C to 30°C) for at least
3 minutes.
9. Use the “RB” specimen to adjust the spectrophotometer or colorimeter to zero
absorbance at 540 nm
10. Read the absorbance values for the standard, controls, and unknowns, and
record the results
11. 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.
Reporting of Results
Report your findings from the Manual Hemoglobin Determination by Spectrophotometer
procedure in the Procedure #4 Report Form or other form supplied by your instructor.
Results should be reported as grams per deciliter (g/dL).
Hemoglobin Calculation (Manual Method)
Hemoglobin (g/dL) = Abs of unknown (Au) × Standard Value
Abs of standard (As)
Example 13-1
The absorbance for a 15.0 g/dL HGB standard is 0.256
The absorbance for the unknown is 0.301.
Unknown (g/dL) 0.301 15.0 g/dL or 1.18 15.0 17.6 g/dL
0.256
Limitations
Sulfhemoglobin, which forms when hydrogen sulfide and hemoglobin are combined,
is toxic when present in excess. It is not measured by the above procedures.
Linearity
Accurate up to 20 g/dL for most reagents, standards, and methodology utilized.
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