CLINICAL LAB SCIENEC

CHAPTER 13: HEMATOLOGY AND COAGULATION 345
Procedure Results
Test Results #1 Test Results #2 Average of #1 and #2 Results
Comments (unusual findings) _____________________________________________________
Performed by: _______________________ Date Reported _________________________
HEMATOLOGY AND COAGULATION PROCEDURE #4
Manual Hemoglobin Determination by Spectrophotometer
Principles
Hemoglobin is a red-pigmented protein that serves to transport oxygen from the lungs
to body tissues and carbon dioxide from the cells of the body back to the heart and to
the lungs to be expelled before picking up more oxygen. Oxygen gives blood a characteristic
bright red color, and venous blood, a deoxygenated state, is darker in color.
Ferricyanide oxidizes oxyhemoglobin to methemoglobin, and cyanide converts
methemoglobin to cyanmethemoglobin. Absorbance measurements are made on a
spectrophotometer at 540 nm. The cyanmethemoglobin reagent contains a surfactant
to promote rapid hemolysis and to accelerate formation of cyanmethemoglobin.
The reaction is completed in less than 3 minutes.
From a clinical perspective, the quantitative determination of cyanmethemoglobin
in the blood is an extremely important procedure for deter-
Safety Alert
mining a number of types of anemia as well as any bleeding that cannot
be seen readily (e.g., internal bleeding). In almost all types of anemia, Hgb
levels are abnormally low and the condition could give evidence of an
underlying disease. Some disease conditions actually have increased Hgb
levels, such as polycythemia, which means “many or much blood.”
The Hgb should compare with the Hct by a 1:3 ratio. If it does not, this
indicates certain abnormalities of the RBCs.
A suitable specimen for a manual hemoglobin determination may be
obtained from a capillary puncture in which a heparinized capillary tube is
used or from whole blood with EDTA used as an anticoagulant; in the latter
case, a plain or nonheparinized capillary tube will be used. Anticoagulants
other than EDTA, such as heparin, citrates, and oxalates, do not maintain the
RBC morphology to the extent that EDTA does but may be used, taking into
account the dilution factor occurring with the use of citrated samples in particular.
Hemoglobin in whole blood collected with EDTA remains stable for 1
week at room temperature (15°C to 30°C) and even longer when refrigerated.
Only a few interfering substances affect the hemoglobin measurement.
Gross lipemia (fatty materials in the blood) will cause elevated Hgb values. Leukocytosis
(abnormally elevated WBC counts) and macroglobulinemia (high levels of large
proteins) may also falsely cause high Hgb values. Although most Hgb determinations
are performed on automated cell counters, it is necessary to perform a manual Hgb
when a sample is lipemic , as the presence of fatty materials in the blood will interfere
Most of these reagents contain
cyanide, an extremely poisonous
substance, but it is used in low
concentrations for this test. The
reagent should not be combined
with a number of chemicals,
including acids. If the reagent
comes in contact with the skin,
the cyanide may be absorbed
through the skin. If the reagent is
ingested, perform gastric lavage
(irrigation of the stomach) and call
a physician. Reagents for disposal
should be discarded by flushing
with copious amounts of water.
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