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Essay On Hemoglobin

Essay on Hemoglobin:- 1.Meaning of Hemoglobin 2. Structure of Hemoglobin 3. Properties 4. Biosynthesis 5. Transportation Provided 6. 2, 3-Biphosphoglycerate (BPG) Stabilizes the T Structure 7. De-oxy-hemoglobin S can Form Fibres that Distort Erythrocytes 8. Varieties 9. Technique for Identification.


  1. Essay on the Meaning of Hemoglobin
  2. Essay on the Structure of Hemoglobin
  3. Essay on the Properties of Hemoglobin
  4. Essay on the Biosynthesis of Hemoglobin
  5. Essay on the Transportation Provided by Hemoglobin
  6. 2, 3-Biphosphoglycerate (BPG) Stabilizes the T Structure of Hemoglobin
  7. De-oxy-hemoglobin S can Form Fibres that Distort Erythrocytes
  8. Varieties of Human Hemoglobin
  9. Essay on the Technique for Identification of Hemoglobins

Essay # 1. Meaning of Hemoglobin:

Hemoglobin is the red colouring matter of blood which is present in the red blood cells. It is a conju­gated protein consisting of heme and the protein globin. It has a molecular weight of 64,450. It can combine with oxygen and acts as the transport mechanism for oxygen within the blood. It con­tains 4 gram atoms of iron per mole in the ferrous (Fe++) state.

Essay # 2. Structure of Hemoglobin:

The structure of Hemoglobin can be classified un­der two headings:

a. Structure of Heme, the prosthetic group.

b. Structure of Globin, the protein part— apoprotein.

a. Structure of Heme:

i. It is an iron porphyrin. The porphyrins are cyclic compounds with “tetra pyrrole” structure.

ii. Four pyrrole rings called I to IV are linked through methylene bridges or methylidene bridges.

iii. The outer carbon atoms, which are not linked with the methylidene bridges, are numbered 1 to 8.

iv. The methylidene bridges are designated as α, β, γ, δ, respectively.

v. Iron in the ferrous state is bound to the nitrogen atom of the pyrrole rings.

vi. Iron is also linked internally (5th linkage) to the nitrogen of the imidazole ring of Histidine of the polypeptide chains.

vii. The propionic acid of 6th and 7th posi­tion of heme of III and IV pyrroles are also linked to the amino acids Arg and Lys of the polypeptide chain, respectively.

The porphyrins are found in nature in which the various side chains are substituted for the 8 hydrogen atoms as numbered in the porphin nu­cleus. The arrangement of the A and P substituents in the uroporphyrin shown here is asymmetric (in ring IV the expected order of the acetate and propi­onate substituents is reversed).

This type of asym­metric substitution is classified as a type III por­phyrin. A porphyrin with a completely symmetri­cal arrangement of the substituents is classified as a type I porphyrin. Only types I and III are found in nature and the type III series is more abundant.

b. Structure of Globin:

i. The globin of hemoglobin is a protein which is composed of 4 parallel layers of closely packed polypeptide chains.

ii. Two of the chains (α-chains) have identi­cal amino acid composition of 141 amino acids. The two other chains may be two of the 4 polypeptide chains designated as β, γ, δ, and ɛ (epsilon). Each is having 146 amino acids.

iii. The total number of amino acids in globin is 574.

iv. α chains have Val-Leu-Ser in N terminal residues and Lys-tyr-Arg in C terminal residues.

v. β chains have Val-His-Leu in N-terminal residues and Lys-tyr-His in C-terminal residues.

vi. γ chains have Gly-His-Phe. N-terminal residues and Arg-Tyr-His in C-terminal residues.

vii. Hemoglobin molecule and its sub-units contain mostly hydrophobic amino acids internally and hydrophilic amino acids on their surfaces. So they form “‘Heme pock­ets”.

viii. In “heme pockets” α subunits are of size necessary for entry of O2 molecule but the entry of O2 molecule in β subunit is blocked by valine residue.

Biosynthesis of Porphyrins:

Chlorophyll (magnesium-containing porphyrin), the photosynthetic pigment of plants and heme (the iron-containing porphyrin) of hemoglobin in ani­mals are synthesized in living cells by a common pathway:

i. The starting materials are ‘active succi­nate’ (succinyl-CoA) derived from the cit­ric acid cycle and glycine. Pyridoxal phos­phate (B6-PO4) is necessary to activate gly­cine. The product of the condensation re­action is α-amino-β-ketoadipic acid which is catalyzed by the enzyme AmLev synthetase (ALA synthase).

ii. α-amino-β-ketoadipic acid is rapidly decarboxylated by the same enzyme AmLev synthetase producing δ-aminolevulinic acid (AmLev). Synthesis of aminolevulinic acid occurs in the mito­chondria. The anemia has been observed in the deficiency of vitamin B(, or pan­tothenic acid.

iii. 2 mols of AmLev condense to form por­phobilinogen (the first precursor of pyrrole) which is catalyzed by the enzyme δ-aminolevulinase (AmLev dehydrase).

iv. 3 mols of porphobilinogen condense first to form a tripyrrylmethane which then breaks down into a di-pyrrylmethane and a monopyrrole. The dipyrryl compounds are of two types A and B. The formation of tetrapyrrole occurs by condensation of two dipyrrylmethanes. If two of the (A) com­ponents condense, a type I porphyrin re­sults; if one (A) and one (B) condense, a type III results.

v. The uroporphyrinogens I and III are con­verted to coproporphyrinogens I and III by decarboxylation being catalyzed by uroporphyrinogen decarboxylase.

vi. The coproporphyrinogen III then enters the mitochondria where it is converted to protoporphyrinogen III and then to pro­toporphyrin III. The enzyme copropor­phyrinogen oxidase catalyzes the forma­tion of protoporphyrinogen III. The oxi­dation of protoporphyrinogen to pro­toporphyrin is catalyzed by the enzyme protoporphyrinogen oxidase.

The enzyme coproporphyrinogen oxidase is able to act on type III coproporphyrinogen only for which type I protoporphyrin has not been identified in natural materials. In mamma­lian liver the reaction of conversion of coproporphyrinogen to protoporphyrin requires molecular oxygen.

vii. In the final step of heme synthesis ferrous ion (Fe++) is incorporated into protopor­phyrin III which is catalyzed by heme syn­thetase or ferrochelatase. The reaction takes place readily in the absence of en­zymes but becomes rapid in presence of enzymes.

A summary of the steps is given:


a. The porphyrinogens are the reduced porphyrins containing 6 extra hydrogen atoms. The oxidized porphyrins cannot be used for heme or chlorophyll synthesis.

b. The porphyrinogens are readily auto-oxidized to the respective porphyrins in pres­ence of light.

Essay # 3. Properties of Hemoglobin

i. Oxy-hemoglobin:

It forms oxy-hemoglo­bin in combination with oxygen. When hemoglobin is exposed to air, it takes up two atoms of oxygen for each atom of fer­rous ion (Fe++) present. Thus, hemoglobin will take up 4 molecules of oxygen. In low oxygen tension, oxy-hemoglobin gives up O2 readily. By this way, blood carries O2 to different parts of the body.

ii. Formation of Carhamino Compound:

It re­acts with CO2 forming carbamino com­pounds.

Hb-NH2 + CO2 → Hb-NH.COOH

iii. Reaction with Carbon Monoxide:

It forms carboxy hemoglobin after reacting with carbon monoxide (CO). Carboxy hemoglobin is stable and prevents the for­mation of oxy-hemoglobin. So inhalation of even small amounts of carbon monox­ide is highly dangerous.

iv. Buffering Action:

One mol of hemoglobin contains 35 histidine residues. Histidine exerts its buffering action through its ba­sic imidazole ring. Hence, hemoglobin plays an important role in regulating the acid-base balance of blood.

v. Formation of Methemoglobin:

Methemoglobin is formed as a result of the oxida­tion of hemoglobin by the mild oxidizing agent, potassium ferricyanide.

The ferrous ion (Fe++) is oxidized to the ferric ion (Fe+++). Methemoglobin cannot carry oxygen in blood.

It is also formed by the action of some drugs. This is found in the blood of some individuals owing to in­born errors of metabolism.

This can be re­duced to hemoglobin by vitamin C which is used in the treatment of methemoglobinemia.

vi. Sulphemoglobin:

It is formed by the ad­ministration of certain drugs. It continues to remain in the blood and cannot be re­converted into hemoglobin.

vii. Cyanomethemoglobin:

It is formed by the addition of cyanide to methemoglobin. It has a bright red colour.

viii. Absorption Spectra:

The different hemo­globin derivatives can be easily identi­fied by this characteristic absorption spec­tra.

(a) Oxy-hemoglobin:

Two bands—one narrow and the other wide in the green region.

(b) Reduced hemoglobin:

One single broad band in the green region.

(c) Carboxy hemoglobin:

Two bands in the green region.

(d) Methemoglobin:

Three bands – one in red and two in the green regions.

(e) Sulphemoglobin:

Three bands simi­lar to methemoglobin.

Essay # 4. Biosynthesis of Hemoglobin

i. The biosynthesis of hemoglobin takes place in the bone marrow in the erythroid cell during its development to erythrocyte.

ii. It starts appearing at stage II (early nor­moblast) and the synthesis is complete when the cell reaches stage IV (late nor­moblast).

iii. Iron in the ferrous state is incorporated into protoporphyrin to form heme.

iv. The heme gets attached to the newly syn­thesized globin to form hemoglobin.

v. The iron of heme is coordinated to 2 imi­dazole nitrogen of histidine at position 38 and 87 in α-chains and 63 & 92 in β-chains.

In nature, the other metal loporphyrins which are compounds of importance in biologic processes are mentioned:

A. Erythrocruorins:

(a) They are iron porphyrinoproteins oc­curring in blood and tissue fluids of some invertebrates.

(b) Their function is corresponding to hemoglobin.

B. Myoglobins:

(a) They are the respiratory pigments oc­curring in the muscle cells of verte­brates and invertebrates.

(b) The purified one has a molecular weight of about 17,000.

(c) They contain only 1 gram atom of iron per mole.

C. Catalases:

(a) They are iron poiphyrin enzymes.

(b) They have been obtained in crystal­line form.

(c) Their molecular weight is about 225,000.

(d) They contain 4 gram atoms of iron per mol.

(e) In plants, their activity is minimal.

D. Tryptophan Pyrrolase:

(a) It is an iron porphyrin protein.

(b) It catalyzes the oxidation of tryp­tophan to formyl kynurenine.

E. Cytochromes:

(a) Cytochromes means the cellular pig­ments because these pigments are widely distributed not only in the tis­sues of higher animals and plants but also in yeast and bacteria.

(b) At first, cytochromes a, b and c were identified and they had been shown to exist in oxidized and reduced forms and their fundamental role is in cel­lular respiration. At present, some thirty cytochromes are known to ex­ist and according to original cytochromes they are designated as a1, a2, a3, c1, c2, c3, c4, c5, b2, b3, b4 etc.

(c) They are iron porphyrins and act as electron transfer agents in oxidation-reduction reactions.

(d) The important example is cytochrome C which has been obtained in the purified form.

(e) Cytochrome C has a molecular weight of about 13,000 and contains 0.43% iron.

(f) The iron porphyrin group of cyto­chrome C is attached to protein more firmly than in the hemoglobin.

(g) Cytochrome C is quite stable to heat and acids.

(h) The reduced form of cytochrome C is not auto-oxidizable.

(i) At physiological pH Ferro cytochrome C does not combine with O2 or CO as does hemoglobin.

(j) The peptide chain of human heart cy­tochrome C contains 104 amino ac­ids, Acetyl glycine is the N-terminal amino acid and glutamic acid the C-terminal amino acid. The two cysteine residues are located at posi­tions 14 and 17 in the peptide chain.

The linkage of iron in heme occurs through the imidazole nitrogen of a histidine residue at position 18 in the peptide chain.

(k) The degree of difference in primary structure among the 13 cytochrome C might be related to the degree of phytogenetic relationship between the species Eg. The cytochrome C of man as compared to that of rhesus monkey differs by only one amino acid of the 104 amino acids. Human cytochrome C differs from that of the dog in 11 amino acid residues, from that of the horse in 12.

(l) The enzymes that catalyse the reac­tions of molecular oxygen are known as oxidases. Cytochrome a3, which is found in heart muscle and other ani­mal tissues is called cytochrome oxi­dase. These oxidases catalyse many reactions in addition to terminal oxi­dation at the electron transport chain. They can carry three general types of reactions e.g., oxygen transfer, mixed function oxidation electron transfer.

Essay # 5. Transportation Provided by Hemoglobin:

Hemoglobin Transports CO2 and Protons to the Lungs after releasing O2 to the Tissues:

i. Hemoglobin can bind CO, directly when oxygen is released and CO, reacts with the amino terminal a-amino groups of the hemoglobin forming a carbamate and re­leasing protons.

The amino terminal is converted from a positive to a negative charge favouring salt bridge formation between the a and P chains.

ii. At the lungs, hemoglobin is oxygenated, being accompanied by expulsion and sub­sequent expiration of CO2. CO2 is absorbed in blood and the carbonic anhydrase in erythrocytes catalyzes the formation of carbonic acid which is rapidly dissociated into bicarbonate and a proton.

A buffering system absorbs these excess protons to avoid the increasing acidity of blood. Hemoglobin binds two protons for every four oxygen molecules. 3. In the lungs, the process is reversed i.e. when oxygen binds to deoxygenated hemoglobin, protons are released and combines with bicarbonate forming car­bonic acid which is exhaled.

Thus, the binding of oxygen forces the exhalation of CO2. This reversible phenomenon is called the Bohr effect. Myoglobin does not ex­hibit Bohr effect.

Essay # 6. 2, 3-Biphosphoglycerate (BPG) Stabilizes the T Structure of Hemoglobin

i. The increased accumulation of 2, 3- biphosphoglycerate is caused by an oxy­gen shortage in peripheral tissues. BPG is formed from 1, 3-biphosphoglycerate in the glycolytic pathway. One molecule of BPG is bound to central cavity formed by all four subunits of hemoglobin.

This cav­ity is of sufficient size for BPG only when hemoglobin is in the T form. BPG is bound by salt bridges between its oxygen atoms and both chains as well as by Lys EF6 and His H21. Thus, BPG stabilizes the T or deoxygenated form of hemoglobin.

ii. Fetal hemoglobin is more weakly bound to BPG because the H21 residue of the Ƴ chain of HbF is Her rather than His and cannot form a salt bridge with BPG. Hence, BPG has a less profound effect on he stabilization of the T form of HbF and is responsible for HbF to have a higher affinity for oxygen than does HbA.

iii. The trigger for the R to T transition of hemoglobin is movement of the iron in and out of the plane of the porphyrin ring.

Essay # 7. De-oxy-hemoglobin S can Form Fibres that Distort Erythrocytes

i. After the de-oxygenation of hemoglobin S the sticky patch can bind to the comple­mentary patch on another deoxygenated HbS molecule. This binding causes po­lymerization of de-oxy-hemoglobin S form­ing long fibrous precipitates. These ex­tend throughout the erythrocyte and me­chanically distort it causing lysis and a good number of secondary clinical effects.

ii. De-oxy-hemoglobin A although contains the receptor sites for the sticky patch present on deoxygenated HbS, the bind­ing of sticky hemoglobin S to de-oxy-hemoglobin A cannot extend the polymer. Because de-oxy-hemoglobin A does not have a sticky patch to enhance binding to another hemoglobin molecule.

Therefore, the binding of de-oxy-hemo­globin A to the R or the T form of hemoglobin S will reject polymerization.

iii. The polymer forms a twisted helical fiber whose cross section contains 14 HbS mol­ecules. These tubular fibres distort the erythrocyte.

Essay # 8. Varieties of Human Hemoglobin

Normal adult hemoglobin or hemoglobin A has a molecular weight of 64,456 and contains two pairs of peptide chains (α & β) of which α chain contains 141 and β chain contains 146 amino acids.

Fetal hemoglobin (F) is present in very small amounts.

All the normal human hemoglobin’s possess a common half-molecule, i.e. a pair of peptide chains (a chains); the other half consists of a pair of differ­ent types of peptide chains, one type for each hemoglobin. Hemoglobin A2 has two δ chains and hemoglobin F has two γ chains; both types of chains contain 136 amino acids and thus are of the same length as the β chain.

Hemoglobin A is repre­sented as α22A hemoglobin A2 as α2A δ2A and hemoglobin F as α22A for describing abnormal hemoglobin. In early embryonic life, a fourth hemoglobin a2Ae2 exists.

Fetal Hemoglobin:

i. Fetal hemoglobin (F) comprises 50 to 90 per cent of the total hemoglobin in the newborn.

ii. It takes up oxygen more readily at low oxygen tensions and releases carbon di­oxide more readily than adult hemoglobin (A).

iii. It is more resistant to denaturation by al­kali and is more susceptible to conversion to methemoglobin by nitrites (contami­nated water).

iv. Hemoglobin F is gradually replaced by hemoglobin A during the first 6 months of extra uterine life.

v. High concentration of hemoglobin F after two years of age occur in various types of anemia, e.g., sickle cell anemia and thalassemia.

Abnormal Hemoglobin’s:

Over one hundred different types of abnormal hemoglobin’s have been described. Some of these are easily differentiated by their electrophoretic mobilities and have given rise to the concept of “molecular disease” which explains that a defec­tive gene (mutant) may direct the formation of a molecule similar to a normal molecule but differ­ing from it in shape, composition and electrical charge.

One amino acid of the normal hemoglobin is replaced by another amino acid, i.e. acidic amino acid is replaced by a basic or a neutral amino acid for the formation of abnormal hemoglobin. The abnormal hemoglobin’s are named in alphabetic order as C, D, E, F, G, H, K, L, M, N, O, P, Q, S etc.

A. Hemoglobin C:

This occurs in the blood of some Negroes in West Africa. The ab­normality is found in the β chain at posi­tion 6, the amino acid glutamic acid is replaced by Lysine. It is characterized by the mild anemia with a tendency to inf­arction.

B. Hemoglobin S:

This appears among the Negroes of Africa. The abnormality occurs in β chain, glutamic acid at position 6 is replaced by valine. Sickle cell anaemia develops and the RBC becomes long and boat-shaped. The blood becomes more viscous which results in reduced blood flow.

C. Hemoglobin F:

HbF is present in fetus and is replaced by adult hemoglobin as the child grows. It is present only in traces in normal adults, it gets hemolysis rap­idly producing a severe anemia called “Thalassemia major”.

D. Hemoglobin M:

There are two types of HbM-HbM (Boston) and HbM I Wate which are of clinical interest. The abnor­mality is found in the α chain, the histi­dine residues in 58 and 87 position are replaced by tyrosine. Abnormal amounts of methemoglobin are found in the blood of persons affected by this condition. This methemoglobin is not reduced to hemoglobin by reducing agents.

E. Hemoglobin D:

This occurs rarely. It ex­ists in two forms – Dα and Dβ. The persons having HbD do not show any clinical signs and symptoms.

Essay # 9. Technique for Identification of Hemoglobins

Finger print technique Ingram developed a tech­nique by which the peptide chains in hemoglobin could be broken down into several smaller peptide fragments by digestion with trypsin. Trypsin splits the peptides only at points where only lysine and arginine occur.

A mixture of smaller peptides were obtained. He then separated this mixture using pa­per electrophoresis technique and paper chroma­tography. The peptides appeared as spots when ninhydrin was sprayed. Thus peptide maps had been prepared for different hemoglobin’s.

Normal cell function depends on a continuous supply of oxygen. As oxygen is consumed during cell metabolism, carbon dioxide is produced. 

A principle function of blood is the delivery of oxygen (O2), present in inspired air, from the lungs to every cell in the body and delivery of carbon dioxide (CO2) from cells to the lungs, for elimination from the body in expired air. 

These vital gas transport functions are dependent on the protein hemoglobin contained in erythrocytes (red blood cells). Each of the 5 × 1010 erythrocytes normally present in 1 mL of blood contains around 280 million hemoglobin molecules.


The hemoglobin (Hb) molecule is roughly spherical and comprises two pairs of dissimilar subunits (FIGURE 1). 

Each of the subunits is a folded polypeptide chain (the globin portion) with a heme group (derived from porphyrin) attached. 

At the center of each heme group is a single atom of iron in the ferrous (Fe2+) state. Thus heme is a metallo-porphyrin, incidentally responsible for the red color of blood.

FIGURE 1: Schematic of oxygenated hemoglobin (HbA) structure

The oxygen-binding site of Hb is the heme pocket present in each of the four polypeptide chains; a single atom of oxygen forms a reversible bond with the ferrous iron at each of these sites, so a molecule of Hb binds four oxygen molecules; the product is oxyhemoglobin (O2Hb). 

The oxygen delivery function of Hb, that is its ability to "pick up" oxygen at the lungs and "release" it to tissue cells is made possible by minute conformational changes in quaternary structure that occur in the hemoglobin molecule and which alter the affinity of the heme pocket for oxygen. Hb has two quaternary structural states: the deoxy state (low oxygen affinity) and the oxy state (high oxygen affinity). 

A range of environmental factors determine the quaternary state of Hb and therefore its relative oxygen affinity. The microenvironment in the lungs favors the oxy-quaternary state, and thus Hb has high affinity for oxygen here. 

By contrast, the microenvironment of the tissues induces the conformational change in Hb structure that reduces its affinity for oxygen, thus allowing oxygen to be released to tissue cells. 


A small amount (up to 20 %) of CO2 is transported from the tissues to the lungs loosely bound to the N-terminal amino acid of the four globin polypeptide units of hemoglobin; the product of this combination is carbaminohemoglobin. However, most CO2 is transported as bicarbonate in blood plasma. 

The erythrocyte conversion of CO2 to bicarbonate, necessary for this mode of CO2 transport, results in the production of hydrogen ions (H+). These hydrogen ions are buffered by deoxygenated hemoglobin.

The role of hemoglobin in transport of oxygen and carbon dioxide is summarized in FIGURES 2a and 2b.

FIGURE 2a: TISSUES O2 diffuses from blood to tissues, CO2 diffuses from tissues to blood

FIGURE 2b: LUNGS CO2 diffuses from blood to lungs, O2 diffuses from lungs to blood

In capillary blood flowing through the tissues oxygen is released from hemoglobin and passes into tissue cells. Carbon dioxide diffuses out of tissue cells into erythrocytes, where the red-cell enzyme carbonic anhydrase enables its reaction with water to form carbonic acid. 

The carbonic acid dissociates to bicarbonate (which passes into the blood plasma) and hydrogen ions, which combine with the now deoxygenated hemoglobin. The blood flows to the lungs, and in the capillaries of the lung alveoli the above pathways are reversed. Bicarbonate enters erythrocytes and here combine with hydrogen ions, released from hemoglobin, to form carbonic acid. 

This dissociates to carbon dioxide and water. The carbon dioxide diffuses from the blood into the alveoli of the lungs and is eliminated in expired air. Meanwhile, oxygen diffuses from the alveoli to capillary blood and combines with hemoglobin.


Although normally present in only trace amounts, there are three species of hemoglobin: methemoglobin (MetHb or Hi), sulfhemoglobin (SHb) and carboxyhemoglobin (COHb) which cannot bind oxygen. 

They are thus functionally deficient, and increased amounts of any of these hemoglobin species, usually the result of exposure to specific drugs or environmental toxins, can seriously compromise oxygen delivery.

A comprehensive account of hemoglobin structure and function is provided in reference [1].

ctHb, the total hemoglobin concentration is typically defined as the sum of oxygenated hemoglobin, deoxygenated hemoglobin, carboxyhemoglobin and methemoglobin. 



The principle reason for measuring ctHb is detection of anemia and assessment of its severity. 

Anemia can be defined as a reduction in the oxygen-carrying capacity of blood due to a reduction in erythrocyte numbers and/or a reduction in ctHb, so that anemia is established if ctHb is below the lower limit of reference (normal) range [2] (TABLE I). The lower the ctHb, the more severe is the anemia.

TABLE I: ctHb reference ranges (Ref 2)

Anemia is not a disease entity, rather a consequence or sign of disease. The reason why ctHb is such a frequently requested blood test is that anemia is a feature of a range of pathologies, many of which are relatively common (Table II). 

Common symptoms, most of which are non-specific, include: pallor, tiredness and lethargy, shortness of breath – particularly on exertion, dizziness and fainting, headaches, constipation and increased pulse rate, palpitations, tachycardia.

TABLE II: Some of the clinical conditions associated with anemia

The absence of these symptoms does not preclude anemia; many mildly anemic individuals remain asymptomatic, particularly if anemia has developed slowly. 


Whilst anemia is characterized by reduced ctHb, a raised ctHb indicates polycythemia. Polycythemia arises as a response to any physiological or pathological condition in which blood contains less oxygen than normal (hypoxemia). 

The body’s response to hypoxemia includes increased erythrocyte production to increase oxygen delivery, and as a consequence ctHb is raised. This so-called secondary polycythemia is part of the physiological adaptation to high altitude and may be a feature of chronic lung disease.

Primary polycythemia is a much less common malignancy of the bone marrow called polycythemia vera, which is characterized by uncontrolled production of all blood cells, including erythrocytes. Polycythemia, whether secondary or primary, is generally much less common than anemia.



The first clinical test of Hb measurement devised more than a century ago [3] involved adding drops of distilled water to a measured volume of blood until its color matched that of an artificial colored standard. 

A later modification [4] involved first saturating blood with coal gas (carbon monoxide) to convert hemoglobin to the more stable carboxyhemoglobin. Modern hemoglobinometry dates from the 1950s, following development of spectrophotometry and the hemiglobincyanide (cynamethemoglobin) method. 

Adaptation of this method and others for use in automated hematology analyzers followed. Over the past two decades advances have focused on development of methods which allow point-of-care testing (POCT) of hemoglobin. 

This section deals first with consideration of some of the methods currently used in the laboratory and then with those POCT methods used outside the laboratory. 


Nearly 40 years after it was first adopted as the reference method for measuring hemoglobin by the International Committee for Standardization in Hematology (ICSH) [5], the hemiglobincyanide (HiCN) test remains the recommended method of the ICSH [6] against which all new ctHb methods are judged and standardized. 

The detailed consideration that follows reflects its continued significance both as a reference and routine laboratory method. 

3.2.1. Test principle

Blood is diluted in a solution containing potassium ferricyanide and potassium cyanide. Potassium ferricyanide oxidizes the iron in heme to the ferric state to form methemoglobin, which is converted to hemiglobincyanide (HiCN) by potassium cyanide. 

HiCN is a stable colored product, which in solution has an absorbance maximum at 540 nm and strictly obeys Beer-Lambert’s law. Absorbance of the diluted sample at 540 nm is compared with absorbance at the same wavelength of a standard HiCN solution whose equivalent hemoglobin concentration is known. 

Most hemoglobin derivatives (oxyhemoglobin, methemoglobin and carboxyhemoglobin, but not sulfhemoglobin) are converted to HiCN and therefore measured by this method. Reagent diluent (modified Drabkin solution) [7]

Potassium ferricyanide (K3Fe(CN)6)200 mg
Potassium cyanide (KCN)50 mg
Dihydrogen potassium phosphate (KH2 PO4)140 mg
Non-ionic detergent (e.g. Triton X-100)1 mL
Above diluted to 1000 mL in distilled water Manual method

25 µl of blood is added to 5.0 mL reagent, mixed and left for 3 minutes. Absorbance is read at 540 nm against a reagent blank. The absorbance of HiCN standard is measured in the same way. ICSH HiCN standard

The major advantage of this method is that there is a standard HiCN solution manufactured and assigned a concentration value according to very precise criteria laid down and reviewed periodically by the International Council for Standardization in Hematology (ICSH) [6]. 

This international standard solution is the primary calibrant for the commercial standard solutions used in clinical laboratories around the world. Thus all those using HiCN standardization are effectively using the same standard, whose value has been scrupulously validated. Interference

Turbidity due to proteins, lipids and cellular matter is a potential problem with spectrophotometric estimation of any blood constituent, including hemoglobin. 

The large dilution (1:251) of sample largely eliminates the problem, but falsely raised ctHb results can occur in patients whose plasma protein concentration is particularly high [8,9,10]. 

Heavily lipemic samples and those containing very high numbers of white cells (leucocytes) can also artefactually raise ctHb by a similar mechanism [11]. Advantages of HiCN

  • International standard – accurate
  • Easily adapted to automated hematology analyzers; thus reproducible (low SD and CV – within batch CV typically < 0.5 %)
  • Well established and thoroughly investigated – ICSH recommended
  • Inexpensive reagent Disadvantages of HiCN

  • Manual method requires accurate pipetting and spectrophotometer
  • Reagent (cyanide) hazardous
  • The above limit its use outside the laboratory
  • Subject to interference from raised lipids, plasma proteins and leucocyte numbers
  • Does not distinguish those hemoglobin derivatives which have no oxygen-carrying capacity (MetHb, COHb, SHb). Thus may overestimate the oxygen-carrying capacity of blood if these are present in abnormal (more than trace) amounts.


3.3.1. Sodium Lauryl Sulphate method

Sodium Lauryl Sulphate (SLS) is a surfactant which both lyses erythrocytes and rapidly forms a complex with the released hemoglobin. The product SLS-MetHb is stable for a few hours and has a characteristic spectrum with maximum absorbance at 539 nm [12]. 

The complex obeys Beer-Lambert’s law so there is precise linear correlation between Hb concentration and absorbance of SLS-MetHb.

The method simply involves mixing 25 µL of blood with 5.0 mL of a 2.08-mmol/L solution of SLS (buffered to pH of 7.2), and reading absorbance at 539 nm. The results of ctHb by the SLS-Hb method have been shown to correlate very closely (r = 0.998) with the reference HiCN method [13]. 

The method has been adapted for automated hematology analyzers and is as reliable in terms of both accuracy and precision as automated HiCN methods [13,14,15]. A major advantage is that the reagent is non-toxic. It is also less prone to interference by lipemia and increased concentration of leukocytes [13]. 

The long-term instability of SDS-MetHb precludes its use as a standard so the method must be calibrated with blood whose ctHb has been determined using the reference HiCN method.

3.3.2. Azide-methemoglobin method

This method is based on conversion of hemoglobin to a stable colored product azide-methemoglobin which has an almost identical absorbance spectrum to that of HiCN [16]. 

The reagent used in this method is very similar to that used in the HiCN reference method with substitution of sodium azide for the more toxic potassium cyanide. As in the HiCN method, hemoglobin is converted to methemoglobin by potassium ferricyanide; azide then forms a complex with methemoglobin.

ctHb results by this method are comparable to results obtained by reference HiCN method; this is an acceptable alternative manual method. The explosive potential of sodium azide, however, prevents its use on automated hematology analyzers [17]. The azide-MetHb reaction has been adapted for POCT hemoglobinometers.


The POCT methods considered here are:

  • Portable hemoglobinometers
  • CO-oximetry – a method utilized in POCT blood gas analyzers
  • WHO color scale

3.4.1. Portable hemoglobinometers

Portable hemoglobinometers like the HemoCue-B allow accurate determination of hemoglobin at the bedside. They are essentially photometers which allow measurement of color intensity of solutions. 

The disposable microcuvette in which these measurements are made also acts as reaction vessel. The reagents necessary for both release of Hb from erythrocytes and conversion of Hb to a stable colored product are present in dried form on the walls of the cuvette. 

All that is required is introduction of a small sample (typically 10 µL) of capillary, venous or arterial blood to the microcuvette and insertion of the microcuvette into the instrument. 

The instrument is factory precalibrated using HiCN standard, and absorbance of the test solution is automatically converted to ctHb. Result is displayed in less than a minute. Advantages of modern hemoglobinometers include

  • Portability
  • Battery or mains operated, can be used anywhere
  • Small sample volume (10 µL) obtained by finger prick
  • Fast (result in 60 seconds)
  • Ease of use – no pipetting
  • Minimal training required by non-laboratory staff
  • Standardized against HiCN – results comparable to those obtained in laboratory
  • Correction for turbidity. In this respect portable hemoglobinometers superior to most ctHb methods [18].  

This technology has been extensively evaluated in a range of settings and most studies [18-24] have confirmed acceptable accuracy and precision when compared with laboratory methods. Disadvantages

Some studies [23,25], however, have raised concern that in the hands of non-laboratory staff results may be less satisfactory. Despite the simplicity of operation these instruments are not immune from operator error, and effective training is essential. 

There is evidence to suggest that results derived from capillary (finger prick) samples are less precise than those derived from well-mixed capillary or venous samples collected into EDTA bottles [25].

3.4.2. CO-oximetry

A CO-oximeter is a specialized spectrophotometer, the name reflecting the original application, which was to measure COHb and MetHb. 

Many modern blood gas analyzers have an incorporated CO-oximeter, allowing the simultaneous estimation of ctHb during blood gas analysis.

The measurement of ctHb by CO-oximetry is based on the fact that hemoglobin and all its derivatives are colored proteins which absorb light at specific wavelengths and thus have a characteristic absorbance spectrum (FIGURE 3). 

Beer-Lambert’s law dictates that absorbance of a single compound is proportional to the concentration of that compound. If the spectral characteristic of each absorbing substance in a solution is known, absorbance readings of the solution at multiple wavelengths can be used to calculate the concentration of each absorbing substance.


In the CO-oximeter absorbance measurements of a hemolyzed blood sample at multiple wavelengths across the range that hemoglobin species absorb light (520-620 nm) are used by the installed software to calculate the concentration of each of the hemoglobin derivatives (HHb, O2Hb, MetHb and COHb). ctHb is the calculated sum of these derivatives.

All that is required from the operator is injection of a well-mixed arterial blood sample into the blood gas analyzer/CO-oximeter. 

The sample, or a portion of it, is automatically pumped to the measuring cuvette of the CO-oximeter, where – by either chemical or physical action – erythrocytes are lysed to release hemoglobin, which is spectroscopically scanned as described above. 

Results are displayed along with blood gas results within a minute or two.

Several studies [26,27,28] have confirmed that ctHb results obtained by CO-oximetry are not clinically significantly different from those derived from reference laboratory methods. CO-oximetry provides an acceptable means of urgent estimation of ctHb in a critical care setting. The particular advantages of ctHb by CO-oximetry include

  • Speed of analysis
  • Ease of analysis
  • Small sample volume
  • No capital or consumable cost beyond that required for blood gas analysis
  • Additional parameters (MetHb, COHb, O2Hb) measured
  • Not affected by high white-cell count [29]

3.4.3. WHO hemoglobin color scale (HCS)

Developed for the World Health Organization (WHO), this low-technology test has limited application in developed countries but has huge significance for the economically deprived countries of the developing world, where anemia is most prevalent. 

In areas where there are no laboratory facilities and insufficient resources to fund more sophisticated POCT hemoglobinometers, it is virtually the only means of determining ctHb.

The HCS test is based on the simple principle that the color of blood is a function of ctHb. A drop of blood is absorbed onto paper and its color compared with a chart of six shades of red, each shade representing an equivalent ctHb: the lightest 40 g/L and the darkest 140 g/L. Although in principle very simple, considerable research and technology was used in development to ensure maximum possible accuracy and precision [30]. 

For example, extensive trials of different papers informed the final choice of paper for the test strip matrix, and spectrophotometric analysis of blood and dye mixtures were employed to arrive at the closest possible match between chart color and the color of blood at each reference ctHb. Advantages of the HCS test

  • Is easy to use – requires only 30 minutes training
  • Requires no equipment or power
  • Is fast – result within 1 minute
  • Requires only a finger prick (capillary) sample
  • Is very cheap (around USD 0.12 per test) Disadvantages of the HCS test

Reliable results depend on strict adherence to test instructions [31].

Common errors include:

  • Inadequate or excessive blood on the test strip
  • Reading result too late (beyond 2 minutes) or too soon (less than 30 sceonds)
  • Reading the result under poor lighting conditions 

The HSC test clearly has inherent limitations [32]. At best it can determine that the ctHb of a patient sample lies within one of six concentration ranges: 30-50 g/L, 50-70 g/L, 70-90 g/L, 90-110 g/L, 110-130 g/L or 130-150 g/L. Still this is theoretically sufficient to identify all but the most mildly anemic patients and give an indication of severity.

An early study [30] demonstrated the test's ability to identify anemia (defined as ctHb < 120 g/L) in 1213 samples with a sensitivity of 91 % and specificity of 86 %. Subsequent trials [31,33] have confirmed that it is an acceptable clinical tool to screen for anemia in the absence of more sophisticated technology and is significantly more sensitive and more specific than clinical examination.


ctHb is one of two parameters routinely used to assess the oxygen-carrying capacity of blood and thereby establish a diagnosis of anemia and polycythemia. 

The alternative test, called the hematocrit (Hct) or Packed Cell Volume (PCV), was the subject of a previous companion article, where the relationship between ctHb and Hct was discussed [34]. The focus of this article has been measurement of ctHb. 

Numerous methods have been devised, the majority based on measuring the color of hemoglobin or a derivative of hemoglobin. For this short review it has inevitably been necessary to be selective. The methods chosen for discussion are among the most commonly used today. 

In making the selection an attempt has been made to convey the range of technologies that are currently employed and how these are applied to satisfy the clinical demand for ctHb in settings that range from impoverished areas of the developing world, where medical care barely has a foothold, to the high-tech world of the modern intensive care unit.