Quantitation of Methemoglobin
Methemoglobin
• Methemoglobin (Hi) is a form of hemoglobin
In which the ferrous ion( Fe2+) has been
oxidized to the ferric state(Fe3+) and is,
therefore, incapable of reversibly combining
with oxygen (and, therefore, cannot transport
the oxygen molecule).
Methemoglobin
• Normally, a small amount of methemoglobin is
continuously being formed in the red cell, but is,
in turn, reduced by the red blood cell enzyme
systems:
1. NADH methemoglobin reductase (cytochromeb5 reductase) (major pathway),
2. NADPH methemoglobin reductase (minor
pathway)
3. and to a lesser extent the ascorbic acid and
glutathione enzyme systems.
Increased amounts
Increased amounts may be found in both hereditary and
acquired disorders
1- The hereditary form of methemoglobinemia is found
A. in disorders in which the red blood cell reducing systems
are abnormal and unable to reduce methemoglobin back
to oxyhemoglobin.
B. in the presence of hemoglobin M, where the structure of
the polypeptide chains making up the hemoglobin
molecule is abnormal (there is a tendency toward
oxidation of hemoglobin, with a decreased ability to
reduce it back to oxyhemoglobin).
2. The acquired causes of methemoglobinemia
are mainly due to certain drugs and
chemicals, such as nitrates, nitrites,
quinones, chlorates, sulfonamides and
aniline dyes.
Reference range
• Methemoglobin is normally present in the
blood in a concentration of 0.03 to 0.13 g/ dL,
but a normal range should be determined by
each laboratory. Slightly higher levels are
present in infants and heavy smokers.
Reagents and Equipment
1.
2.
3.
4.
5.
6.
Phosphate buffer, 0.017 M (pH 6.6)
Neutralized sodium cyanide.
Potassium ferricyanide, 20% w/v .
Test tubes.
Pipets, 1 mL, 10-100 µL .
Spectrophotometer.
Specimen
• Fresh anticoagulated whole blood, using EDTA
or heparin as the anticoagulant.
• This test should be perform d within 1 hour of
blood collection. However, once the blood has
been diluted in the buffer reagent, it may be
stored at 2 to 6° C for a maximum of 24 hours.
Principle
• Whole blood is diluted with a phosphate buffer solution.
Methemoglobin has a maximum absorbance at a
wavelength of 630 nm.
• The diluted specimen is read in a spectrophotometer at 630
nm and the absorbance reading noted (D1). Neutralized
sodium cyanide is added to the mixture, converting the
methemoglobin to cyanmethemoglobin, and read on the
spectrophotometer (D2).
• The change in optical density is directly proportional to the
amount of methemoglobin present. The methemoglobin in
g/dL is then calculated using a factor, previously
determined, for the spectrophotometer used.
Procedure
1- Determination of calculation factor
– Before the methemoglobin results can be calculated, a
calibration factor for the spectrophotometer being used must be
determined. This is done one time only, or whenever a different
spectrophotometer is used for the procedure.
– Obtain a whole blood sample and determine the hemoglobin
concentration in g/dL .
– Place 5.0 mL of 0.017 M phosphate buffer into each of two test
tubes. Add 50 µL of the whole blood specimen to one tube and
mix. The second tube is to be used as the blank.
Procedure
– Add 20 µL of freshly prepared 20% potassium ferricyanide to
both tubes. Mix and allow to stand for 2 minutes.
– Read on the spectrophotometer at 630 nm, using the blank to
set the optical density at 0, and record the absorbance ( Dx).
– Add 20µLof neutralized sodium cyanide to both tubes. Mix and
allow to standfor 2 minutes. Read on the spectrophotometer as
above and record the optical density ( Dy).
• F =Hgb (g/dL)/Dx - Dy
Procedure
2. Procedure for testing patient specimen
– Label and place 5 mL of 0.0 17M phosphate buffer
into one tube for each specimen and control to be
tested and one additional tube to serve as a
blank .
– Add 50 µL of whole blood to the appropriately
labeled tube and mix well. Allow to sit at room
temperature for 5 minutes.
Procedure
– Read on the spectrophotometer at a wavelength
of 630 nm, using the blank to set the optical
density at 0. Record the absorbance of the
solution (D1)
– Add 20 µL of neutralized sodium cyanide reagent
to each tube including the blank, and mix. Allow
to sit for 2 minutes. Read as in step c above record
the optical density (D2).
Calculation of results
Methemoglobin (g/dL) = ( D1 – D2) x F
Normal value
Methaemoglobin<2% of total haemoglobin in normal
blood, slightly higher in infants, particularly if
premature
Discussion
• Sulfhemoglobin (SHb ) is not measured at any
time during the above procedures.
• The presence of a large amount of SHb will
result in an erroneously low measurement of
total Hb.