HAEMOGLOBIN

Hemoglobin or haemoglobin (Hb) is the iron-containing oxygen-

transport metalloprotein in the red cells of the blood in mammals and other
animals. Hemoglobin in vertebrates transports oxygen from the lungs to the rest of
the body, such as to the muscles, where it releases the oxygen load. Hemoglobin
also has a variety of other gas-transport and effect-modulation duties, which vary
from species to species, and which in invertebrates may be quite diverse.

The name hemoglobin is the combination of heme and globin, reflecting the fact
that each subunit of hemoglobin is a globular protein with an embedded heme (or
haem) group; each heme group contains an iron atom, and this is responsible for
the binding of oxygen. The most common types of hemoglobin contains four such
subunits, each with one heme group.

Types of Haemoglobins in humans

In the embryo:

• Gower 1 (ξ2ε2)

• Gower 2 (α2ε2)

• Hemoglobin Portland (ξ2γ2)

In the fetus:

• Hemoglobin F (α2γ2)

In adults:

• Hemoglobin A (α2β2) - The most common type.

• Hemaglobin A2 (α2δ2) - δ chain synthesis begins late in the third trimester

and in adults, it has a normal level of 2.5%

Normal Values:
Normal results vary, but in general are:

• Male: 13.8 to 17.2 gm/dL

• Female: 12.1 to 15.1 gm/dL

FUNCTIONS OF HAEMOGLOBIN AND RBCs:

• They help to carry O2 and CO2 from lungs to the tissues and vice versa.

• Haemoglobin due to the buffering action helps to maintain the acid base balance of the
body.

• RBCs due to selective permeability of their cell membrane help to maintain the ion
balance.

• RBCs are responsible for maintaining the viscosity of whole blood.

• Various pigments of bile like bilirubin and biliverdin are formed from haemoglobin.

Anemia
Decreased RBC count or decreased hemoglobin level in RBCs below normal, is
called anemia.

TYPES OF ANEMIA:

A) Nutritional Anemia:

1) Iron deficiency anemia:

Also called microcytic hypochromic or blood loss anemia.

Causes:

a) Chronic blood loss (hemorrhage), when a person cannot absorb

enough iron from GIT to form hemoglobin as rapidly as it is lost,
so RBCs are small sized and contain little Hb.

b) Transferrin deficiency; Iron cannot be transported from GIT to

erythroblasts to form Hb, so RBCs contain little Hb.

2) Pernicious Anemia:

Also called megaloblastic or macrocytic anemia.

Causes:

a) Lack of Vit. B12 c) Lack of intrinsic

factor
b) Lack of folic acid
B) Aplastic Anemia:

In this type of anemia RBC count is decreased due to bone marrow aplasia or
lack of functioning bone marrow.

Causes:

Bone marrow aplasia or destruction by:

a) Gamma rays c) Chemicals

b) X-rays d) Drugs

C) Hemolytic Anemia:

In these types of anemia RBCs rupture with release of Hb.

Types:

1) Hereditary Spherocytosis:

RBCs are small and spherical rather than biconcave discs. RBCs donot have
normal, loose, bag like membrane and cannot be compressed, so they
rupture on passing through splenic pulp.

2) Sickle Cell Anemia:

RBCs contain abnormal HB S, which precipitates into long crystals in low

oxygen tension, which give sickle shape to RBCs. Hb precipitate damages cell
membrane and makes it fragile, so the RBCs rupture on passing through
small capillaries.

3) G6PD Deficiency Anemia:

Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency is a hereditary

condition in which red blood cells break down when the body is exposed to
certain drugs or the stress of infection.

4) Thalassemia:

It is an inherited autosomal recessive disorder, in which RBCs are unable to

synthesize alpha or beta polypeptide globin chains that form hemoglobin.
Reduced synthesis of one of the globin chains causes the formation of
abnormal hemoglobin molecules and this in turn causes the anemia.

Symptoms of Anemia:
 • Fatigue

• Headaches

• Faintness

• Breathlessness

• Angina

• Intermittent claudication

• Palpitations.

Signs of Anemia:
• Pallor

• Tachycardia

• Systolic flow murmur

• Cardiac Failure

HAEMOGLOBIN ESTIMATION
PRINCIPLE:
Haemoglobin (Hb) in the sample, in the presence of ferricyanide, is oxidized to
methaemoglobin (Hi), which, then reacts with cyanide at pH 7.2, producing
haemoglobin cyanide (HiCN or cyanmethaemoglobin).

All hemochromogens, except for sulfhaemoglobin, completely react in 3 minutes

and the reading is performed at 540 nm.

PROVIDED REAGENTS:

Surfactant/ CNX: Breakable ampoules containing stabilized solution of

surfactant/CNX.

Buffer/ ferricyanide: Stabilized tablets of potassium ferricyanide and phosphate

buffer.

Final concentrations:

• Potassium ferricyanide____________600 µmol/l

• Cyanide________________________768 µmol/l
• Phosphates______________________ 1 µmol/l
• Non-ionic surfactant_______________0.5 µmol/l
• pH_____________________________ 7.2 + 0.1

STANDARD:

Wiener lab’s haemogloWiener standard

INSTRUCTIONS FOR USE:

Pour contents of one surfactant/ CNX ampoule into one liter cylinder, avoiding any
loss or spilling. Add 700-800 ml of distilled water and one buffer/ferricyanide tablet.
Mix by swirling until complete dissolution and bring to volume with distilled water.
Transfer to amber glass bottle, label and date.

WARNINGS:

• Surfactant/ CNX contain cyanide (poison). Take necessary care for

handling, following the sample procedure with cyanide containing waste
avoiding any contact with acids in sinks or pipelines.
• Reagents are for in vitro diagnostic use.
• Never pipette out by mouth, always use pipette sucker.

STABILITY AND STORAGE INSTRUCTIONS:

• Provided reagents: Stable at room temperature until expiration date.

 • HaemogloWiener reagent: Stable 6 months since preparation date, at
room temperature and thoroughly protected from light. Do not freeze or
refrigerate.

SAMPLE:

• Collection: Collect anticoagulated blood.

• Additives: Use concentrated EDTA anticoagulants or dried balance
anticoagulants.
• Stability and storage instructions: blood haemoglobin contents are
stable up to one week in refrigerator (2-10 0C). Do not freeze.

REQUIRED MATERIALS

• Spectrophotometer.
• Micropipettes and pipettes for measuring the stated volumes.
• Spectrophotometer cuvettes or Photo colorimeter tubes.
• Watch or timer.
• Graduated flask.
• Amber glass bottle.

ASSAY CONDITIONS:

• Wave-length: 540nm in spectrophotometer, 520-550nm for photo

colorimeter with green filter.
• Reaction temperature: Room temperature.
• Reaction time: 3 min.
• Sample volume: 20 µl.
• Reagent volume: 5 ml.
• Final reaction volume: 5.02 ml

PROCEDURE:

B S U

Standard - 20 µl -

Sample - - 20 µl

Reagent 5ml 5ml 5ml

STABILITY OF FINAL REACTION:

 Final reaction color is stable for at least 24 hours, thus readings may be
performed within this period.

OBSERVATIONS AND CALCULATIONS:

O.D. of sample: ____________

O.D. of standard: ___________

Concentration of standard: ___15.6g/dl______

Hemoglobin (g/dl) = O.D of sample × Conc. Of Standard

------------------

O.D. of Standard

Haemoglobin = _____________g/dl.