Hemoglobin is an iron–bearing
protein contained within the erythrocytes. It is a respiratory protein which
serves as the main component of red blood cells. It is a conjugated protein
that serves as the vehicle for the transportation of oxygen and carbon dioxide.
It is a chromoprotein consisting of 35% solid material in the red cells. A
chromoprotein is a compound of a protein molecule and a non–protein pigment.
Structure of
hemoglobin
Hemoglobin is composed of
one molecule of globin, a basic protein and four molecules of heme, an organic
compound of iron. It has an approximate molecular weight of 68,000. It is
believed to be elliptical in shape.
The molecule of hemoglobin
is composed of two identical half–molecules, each containing two different
peptide chains, designated alpha and beta. The alpha consists of 141 amino acid residues
and the beta chain, 146 amino acid residues. The precise organization of these
amino acids is vital to a normal molecule, and one substitution along the amino
acid chain known to alter the hemoglobin function and result in a group of
disease called “hemoglobinopathies.”
Formation of
hemoglobin
Synthesis of hemoglobin
begins in the erythroblast and continuous throughout the normoblastic stage.
Even when young RBC leaves the bone marrow and pass into the blood stream they
continue to form hemoglobin for a few days. Thus, hemoglobin formation does not
depend upon a specific structure of the bone marrow but, instead, is an
intrinsic ability of the early red blood cells themselves.
From tracer studies with
isotopes, it is known that hemoglobin is synthesized mainly form acetic acid
and glycine. It is believed that acetic acid is changed into alpha–glutaric
acid, and then 2 molecules of this combine with one molecule of glycine to form
a pyrrole compound. In turn, 4 pyrrole compounds combine to form a
protoporphyrin compound. One of the protoporphyrin compound, known as as the
protoporphyrin III, then combines with iron to form the heme molecule. Finally,
4 heme molecules combine with one molecule of globin to form hemoglobin
A.
1
alpha–ketoglutaric acid + glycine -------> pyrrole
B.
4 pyrrole
---------> protophorphyrin
C.
Protoporphyrin
III + Fe ------------> heme
D.
4 heme + globin
------> hemoglobin
Accessory substances
needed for formation of hemoglobin
In addition to amino acid
and iron, which are needed directly for formation of hemoglobin molecule, a
number of other substances act as catalyst or enzymes during different stages
of hemoglobin formation. These are:
a. Copper
b. Pyridoxine
c. Cobalt
d. Nickel
The above mentioned
substances serve mainly to emphasize the fact that hemoglobin formation results
from a series of synthesis reactions, each of which depends upon appropriate
building materials and also upon appropriate controlling catalyst and enzymes.
Function of
hemoglobin:
1. Transport oxygen
to body tissues.
2. Removes carbon
dioxide, a product of metabolism, from the body tissues
3. Act as the most
important buffer in the blood
Normal hemoglobin:
The heme group is
identical in all variants of human hemoglobin. The protein part of the molecule
(globin) consists of four polypeptide chains. At least three distinct
hemoglobin types are found postnatally in normal individuals, and the structure
of each has been determined.
1. Hemoglobin
A1 (HbA1) – the
major normal adult hemoglobin present in more than 95% of normal adults. The
polypeptide chains of the globin part of the molecules are
Two
identical alpha chains
Two
identical beta chains
2. Hemoglobin
A2 (HbA2) – a
normal adult hemoglobin which constitutes less than 35% of the total
hemoglobin. The polypeptide chains are:
Two
alpha chains
Two
beta chains
3. Hemoglobin
A3 (HbA3) – a
degradation product of HbA2 which comprises of 5 – 15% of the total adult
hemoglobin. The polypeptide chains are:
Two
alpha chains
Two
beta chains
4. Embryonic
hemoglobin – these are found in
normal human embryos and fetuses with a gestational age of less than three
months:
Hb
Gower – 1 – composed
of 2 zeta and 2 epsilon chain
Hb
Gower – 2 – composed of 2 alpha and 2 epsilon chains
Hb
Portland – 1 – composed of 2 zeta and 2 gamma chains
5. Fetal
hemoglobin – HbF is the major Hb of
the fetus and newborns. The polypeptide chains are: 2 alpha and 2 gamma chains
Reactions of normal
hemoglobin
After traveling through
the pulmonary arteries in the lungs, the blood becomes oxygenated in the
capillaries, where the oxygen in the lung diffuses into the blood. Oxygenated
hemoglobin is referred to as oxyhemoglobin (HbO2). The binding of
the oxygen to the hemoglobin molecule is rather loose compared to other more
stable chemical bonds.
After hemoglobin loses its
oxygen to the various tissue of the body, it become known as reduced hemoglobin
Oxyhemoglobin – by virtue of its iron, oxyhemoglobin, a compound
that gives the bright red color to oxygenated blood in the arteries. It is the
presence of oxyhemoglobin passing through capillaries near the body surface
that gives the pinkness to the skin and varying degrees of redness to mucous
membranes and the lips.
Reduced hemoglobin – is hemoglobin that has given up its oxygen and is
blue in color rather than red. Under normal conditions not enough reduced
hemoglobin is formed for the blue color to be visible, and blood in the
systemic veins merely appears dark red in color. However, various diseases or
abnormalities related to the cardiovascular and respiratory systems may result
in the presence of such large quantities of reduced hemoglobin in the
capillaries that the skin and mucous membrane have a distinctly blue color,
this condition is called cyanosis.
Hemoglobin
derivatives
The two physiologic hemoglobins, the oxyhemoglobin and the reduced hemoglobin, are readily converted to a series of compounds through the action of acids, alkalies, oxidizing and reducing substances, heat and other agents.
1. Hemiglobin
(Hi) or Methemoglobin (MHb) or Ferrihemoglobin
Hi
is a derivative of hemoglobin in which the ferrous ion is oxidized to the
ferric state. This abnormal form of oxidized Hb is chocolate brown in color.
2. Sulfhemoglobin
In
vitro and in the presence of oxygen, hemoglobin reacts with hydrogen sulfide to
form a greenish derivative of hemoglobin called sulfhemoglobin. It may form in
response to an oxidant stress; further change can result in denaturation and
precipitation of hemoglobin as Heinz bodies.
SHb
cannot transport oxygen, but it can combine with CO2 to form
carboxyhemoglobin (HbCO).
3. Carboxyhemoglobin
Hemoglobin
which combines with carbon monoxide results in the formation of brilliant
cherry red colored carboxyhemoglobin (HbCO). The chief sources of CO gas are
gasoline moors, illuminating gas, gas heaters, defective stoves and the smoking
tobacco.
Abnormal hemoglobin
and nomenclature
Abnormal hemoglobin is
formed if there is a permanent structural rearrangement of molecular
composition of the hemoglobin. The abnormal hemoglobin may or may not produce a
disease state. When it is associated with a disease process, the condition is
termed hemoglobinopathy.
1. Hemoglobin
Bart’s – an abnormal variant of HbF
2. Hemoglobin
H – the hemoglobin that moves fastest
toward the anode in hemoglobin electrophoresis. It is often found in
association with an alpha chain disorder and with thalassemia minor.
3. Hemoglobin
C – abnormal Hb wherein the
substitution of amino acid is in the sixth position of the beta chain.
4. Other
hemoglobin was assigned letters of the alphabet, then geographical names, or
both.
The sequence of eight
amino acids in the Beta – peptide chain of Hb showing the substitution
abnormality in hemoglobin S and C:
Amino Acid Sequence
Hb
|
A
|
S
|
C
|
1
|
Valine
|
Valine
|
Valine
|
2
|
Histidine
|
Histidine
|
Histidine
|
3
|
Leucine
|
Leucine
|
Leucine
|
4
|
Threonine
|
Threonine
|
Threonine
|
5
|
Proline
|
Proline
|
Proline
|
6
|
Glutamic
acid
|
Valine
|
Lysine
|
7
|
Glutamic acid
|
Glutamic acid
|
Glutamic acid
|
8
|
Lysine
|
Lysine
|
Lysine
|
Destruction of
hemoglobin
When a red cell fragments,
its hemoglobin is immediately released into the plasma. Very soon, this
phagocytized by the reticuloendothelial cells, which split the heme portion
from the globin molecule. The result is a straight chain of four pyrrole nuclei,
which is the basic structure of bile pigments. The first pigment formed is
biliverdin, but this is rapidly reduced to bilirubin. These products are
gradually released into the plasma.
Bilirubin is insoluble in
water, but it combines firmly with plasma proteins in which form it is soluble
and is transported throughout the body. On reaching the liver, the liver cells
remove bilirubin from the protein and conjugate approximately 80% of it with
glucuronic acid to form bilirubin glucuronide. This is highly soluble in water
and is normally secreted by the liver cells into the bile. An additional 10% is
conjugated with sulfate to form the soluble bilirubin sulfate, and the final
10% is conjugated with other solubilizing substances, all of which are
similarly excreted.
It is bilirubin in the
bile that gives it its greenish yellow color and any failure of the liver to
excrete bile causes increased quantities of bilirubin in the body fluids. These
in turn produce a yellow color (jaundice) in the skin. Obviously, the more
rapid the destruction of RBC, the greater also will be the amount of bilirubin
in the body fluids.
In forming the bile
pigments, the reticuloendothelial cells remove iron from heme and this is
immediately released into the iron pool of the body. The iron can then be
reused for formation of additional Hb or other substances.
*********
Hemoglobinometry – the measurement of the concentration of hemoglobin
in blood.
The hemoglobin content of
a solution may be estimated by:
a. Measurement of
its color through the color of the blood.
b. A method based on
specific gravity of the blood.
c. By its power of
combining with oxygen
e. By converting
hemoglobin into one of several compounds and comparing the resulting compound
with a known standard either visually or photoelectrically.
Different methods of
hemoglobin determination
1. Specific
gravity method or Copper Sulfate method
In
this method, drop of blood are allowed to fall into 16 small bottles containing
copper sulfate solution of increasing specific gravity readings.
a. If the drop of blood falls in a few seconds, it has a
greater specific gravity than the solution
b. If the drop of blood rises in a few second, it has a
lower specific gravity than the solution
c. If the drop of blood remains suspended for about 15
seconds and then falls, more or less it has the same specific gravity as the
solution
Note: This method is used by blood banks as a
screening test for blood donors.
2. Gasometric
method
Oxygen
capacity method
Principle:
Hemoglobin will combine with and
liberate a fixed quantity of oxygen.The blood is hemolyzed
with saponin and the oxygen is collected and measured in a Van Slyke
apparatus.
Note:
The oxygen combining capacity of blood is
1.34 ml O2 per gram of hemoglobin. The
volume of oxygen is corrected for temperature and pressure and the hemoglobin
concentration is determined with the use of the following formula:
Volume
of oxygen / 100 ml blood = Grams
of Hb/100 ml blood
1.34
Grams
of Hb/100 ml blood x 10 = Grams of Hb / liter of blood
Note: This
method was formerly used to calibrate or standardize instruments for hemoglobin
determination
3. Chemical
method
Hemoglobin
may be measured by determining the iron content of whole blood. Based on the
molecular structure, the iron content of hemoglobin is 0.347%. Thus, 1 gram or
1000 mg of Hb contains 3.47 mg or iron. The concentration of hemoglobin in
blood is calculated by dividing the iron content (mg/dl) by 3.47
Wong’s
method
Principle:
Iron
is detached from the hemoglobin by treating the blood with concentrated sulfuric
acid in the presence of potassium persulfate. The protein are precipitated with
tungstic acid and filtered off. The iron content of the filtrate is determined
in a colorimeter and the Hb value is calculated with the following formula:
mg
iron / 100 ml = grams
of Hb / 100 ml blood
3.47
Grams
of Hb / 100 ml x 10 = grams of Hb / liter
Note: This method has been used for calibrating
various hemoglobinometers
4.
Colorimetric
method
a. Visual
colorimetric method
(1)
Direct matching
method
Principle:
The
color of fresh blood is compared with a series of colored standards representing
known quantities of hemoglobin.
The
procedure employed in the following are based on the principle of direct
matching method:
(a) Tallquist method
(b) Dare’s method
(c) Spencer’s method
(2)
Acid hematin
method
Principle:
Blood
is mixed with 0.1 N HCl. This hemolyzes the red cells and converts the
hemoglobin to a brownish yellow solution of acid hematin. The acid hematin is
then compared with a colored glass standard (Comparator Block)
The
procedures employed in the following are based on the principle of Acid Hematin
method:
(a)
Sahli – Hellige
method
(b)
Haden – Hausser
method
(c)
Sahli – Adams
method
(d)
Osgood – Haskin
method
(e)
Haldane method
(f)
Newcomer method
(3)
Alkali
hematin method
Principle:
Blood
is mixed with 0.1 N NaOH. The solution is then boiled. The hemoglobin is then
converted to a blue–green solution of alkaline hematin. The color of the
solution is then compared with a known standard or in a colorimeter
Note:
This
method will not accurately measure the hemoglobin of an infant, because
infant’s blood contains alkali resistant fetal hemoglobin (HbF)
The
principle of Alkali – hematin method is used in the following:
(a) Standard method using Gibson and Harrison’s standard
solution
(b) Clegg and King method
b.
Photoelectric
method
(1)
Oxyhemoglobin
method
Principle:
Blood
is mixed with either 0.1% sodium carbonate or 0.007 N Ammonium hydroxide
solution. This converts the Hb to oxyhemoglobin. The depth of the resulting
color is then measured in a photometer with a green filter (540 nm) and 0.007 N
ammonium hydroxide as a blank
(2)
Cyanmethemoglobin
method (MHbCN method) or Hemiglobincyanide (HiCN) method
Principle:
Blood
is diluted with Drabkin’s solution which contains ferricyanide and Cyanide. The
potassium ferricyanide oxidizes hemoglobin to hemiglobin and potassium cyanide
provides cyanide ions to form hemiglobincyanide, which has a broad absorption
maximum at a wavelength of 540 nm. The absorbance of the solution is measured
in a Photometer or spectrophotometer at 540 nm and compared with that of a
standard HiCN solution.
Absorbance
of test sample x Concentration
of standard (mg/dl) x 251
Absorbance
of standard 100 mg
= Hb
(g/dl)
Drabkin’s
reagent
Potassium
ferricyanide – 0.200 g
Potassium
cyanide – 0.050 g
Dihydrogen
potassium phosphate – 0.140 g
Non–ionic
detergent
Storox –
0.5 ml
Tritron – 1.0 ml
Distilled
water q.s.ad. – 1000 ml
The
solution should be clear and pale yellow, have a pH of 7.0 to 7.4 and give a
reading of zero when measured in the photometer at 540 nm against water blank.
This reagent should be kept in ambered colored bottle.
Note:
The
HiCn method is the method of choice, being considered as the most accurate and
reliable method.
a. Most forms of
hemoglobin (Hb, HbO2, Hi and HbCO2, but not SHb) are
measured
b. The test sample
can be directly compared with HiCN standard
c. Readings can be
made at the convenience of the operator because of the stability of the diluted
samples. The solution of HiCN is the most stable among the hemoglobin
derivatives.
Errors in
hemoglobinometry
1. Errors
inherent in the sample
a. Blood sample collected through improper venipuncture
b. Blood sample collected through improper skin or
capillary puncture technique
2. Errors
inherent in the method
3. Errors
inherent in the equipment
a. The accuracy of
the equipment is not uniform
b. Unmatched
cuvettes
c. Improper
standardization of the photometer or colorimeter
4. Operator’s
error – the so called human errors
Human
errors can be reduced by good training, understanding the clinical significance
of the test and the necessity for a dependable method, adherence to oral and
written instructions and familiarity with the equipment and with the sources of
error. The technologist who is interested in the work will be less prone to
make errors than others.
Normal values
Conventional unit S.I. unit
Men 14 – 18 g / 100 ml blood 140 – 180 g/l
Women 12 – 16 g / 100 ml blood 120 – 160 g/l
Children 14 – 26 g / 100 ml blood 140 – 260 g/l
Test for hemoglobin
derivatives and abnormal hemoglobin pigments
1. Shaking
(naked eye examination)
Shaking
of normal whole blood in the air for 15 minutes imparts to it a bright red
color as the Hb is converted to HbO. The blood is cherry red when the pigment
is HbCO in carbon monoxide poisoning. The color is chocolate brown in
methemoglobinemia (HiCN) and mauve lavender in sulfhemoglobinemia
2. Spectrophotometric
identification of hemoglobin
The
various hemoglobins have characteristic absorption spectra, which can easily be
determined with a spectrophotometer
3. Katayama’s
test – the patient’s blood is mixed
with fresh orange colored ammonium sulfide and then viewed with a hand
spectroscope. Then the spectra of the unknown and the known pigments may be
compared in the spectroscope.
4. Examination
of plasma or serum for methemalbumin
Methemalbumin
is also known as the Fairley’s pigment. In this protein, the ferrous iron is
oxidized to ferric state and is bound to albumin.
Schumm’s
test:
The
plasma or serum is covered with a layer of ether and mixed saturated yellow
ammonium sulfide. The mixture is then viewed with a hand spectroscope. If
methemalbumin is present, a relatively intense narrow absorption band will be
seen in the green filter at 558 nm.
5. Hemoglobin
electrophoresis – this the single
most useful laboratory test for detection and identification of abnormal
hemoglobin.
Hemoglobin
molecule in an alkaline solution have a net negative charge and move toward the
anode in an electrophoretic system at a speed proportional to the strength of
their charge. Those with electrophoretic mobility greater than that of HbA at
pH 8.6 in barbital buffer are known as “fast hemoglobin” and these include Hb
Barts and the two fastest, HbH and Hb1. HbC is the slowest of the common
hemoglobins.
6. Determination
of fetal hemoglobin
a. Alkali
Denaturation Test
Singer
and Chernoff’s method
Fetal
hemoglobin resists alkali denaturation, while adult hemoglobin does not. A
hemolysate is alkalinized and then neutralized and the denatured adult Hb is
precipitated by ammonium sulfate. A filtrate will then contain only alkali resistant
hemoglobin, which is measured and expressed as a percentage of the total.
Normal
values: Adults 0.5 – 0.8%
1 year old 1%
HbF
is increased in thalassemia major, sickle cell disease, and in hereditary
persistence of fetal hemoglobin.
b. Acid – Elution
Test
Kleihauer,
Braun and Betke method
The
identification of cells containing HbF depends upon the fact that they resist
acid–elution to a greater extent than do normal cells, thus, they appear as
isolated darkly–staining cells among a background of pale–staining ghost
cells. Hemoglobin other than HbF are eluted from the red cells on air dried
blood film by citric acid phosphate buffer (pH 3.3)
7. Test to
detect the presence of HbS (Test for Sickling)
For
details, please refer to the topic on sickle cell anemia under the Chapter –
Red Cell Disorders
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