ANTICOAGULANTS

These are substances either in liquid or powder form that prevent blood from clotting
when mixed together.
Prevention of blood from clotting is achieved through transferring the blood sample
into bottles having a known concentration and type of anticoagulant.
Various types of anticoagulants include
1. EDTA- Ethylene diamine tetra acetic acid (sequestrene)
2. Trisodium- citrate
3. Heparin
4. Oxalates
5. Fluoride

1. EDTA(SEQUESTRENE)
This is a dipotassium and disodium salt of Ethylene Diamine tetra acetic acid.
Prevents coagulation by combining with calcium ions thus acting as a chelating
agent.
Used at a concentration of 1.5mg/ml
ADVANTAGES
It is the anticoagulant of choice when total platelets count or concentrates for
transfusion are required, as it prevents them from clumping together.
It can also be used for most routine blood work such as: -cell counts (platelets, red
blood cells) -packed cell volume
-different counts
It does not color blood smears
Hb concentration is stable for 2-3 days at 4-8˚c provided there is no haemolysis
Blood cell counts, reticulocyte count and PCV change very little in EDTA blood
at 4-8˚c when stored for up to 24 hours.
LIMITATAIONS
Unsuitable for coagulation studies since it does not preserve coagulation factors.
It’s prothrombin itself
Unsuitable for osmotic fragility test since it increases osmotic pressure
EDTA is the anticoagulant of choice for routine hematology work, however the
following should be taken care of:
1) When EDTA anticoagulated blood cannot be tested within 1-2 hours it must
be refrigerated at 4-8˚c to prevent cellular changes affecting test results
2) Some of the cellular changes are:
i. Neutrophil degeneration where the neutrophil acquires an irregular
shape, its nuclear lobes separate, vacuoles appear in the cytoplasm and
there is loss of granules.
ii. There is segmentation of budding of the nucleus of lymphocytes and
monocytes and vacuoles appearing in the cytoplasm
 iii. Erythrocytes become crenated and spherocytic
iv. Platelets disintegration
Due to these cellular changes it is advised blood films should be prepared as soon as
possible after blood collection.
In EDTA anticoagulant morphological blood cell changes occur soon after blood is
collected when it is stored at room temperature and within 3 hours when stored at 4-
8˚c and therefore the recommendation of fixing blood films in methanol as soon as
possible after blood collection.
MODE OF PREPARATION
Take 10gms of powder in 100mls of distilled water, then distribute 1.5mls of solution
into anticoagulant bottles them dry.
2. SODIUM CITRATE
Mode of action is by combining with calcium hence preventing prothrombin
conversion, thus preventing coagulation
It is used in liquid state
Anticoagulant of choice in coagulation studies, Westergren ESR and blood
transfusion work, for coagulation studies, factor v is relatively stable in citrated
blood.
Used in proportion 9 volumes of blood to 1 volume of anticoagulant (9:1)
Used in 4 volumes of blood to one volume of anticoagulant at a concentration of
3.8% for Westergren ESR.
3. OXALATES
Commonly used oxalates are potassium, sodium and Ammonium
A mixture of potassium and Ammonium or sodium and ammonium are used
Never use a mixture of potassium and sodium because of electrolyte shift
They act by precipitating calcium as insoluble calcium oxalates thus preventing
clotting
ADVANTAGES
i. Cheap
ii. Easy to prepare
iii. Requires no dilution
USE
Hemoglobin estimation and blood cell counts
LIMITATIONS
i. It is poisonous and hence cannot be used for blood transfusion purposes
ii. Being a salt, it cannot be used for osmotic fragility test due to increase in
osmotic pressure
 iii. It leads to nuclear degeneration of white blood cells as well as crenation of red
blood cells and therefore blood films prepared from such blood have to be
prepared within a short time of blood collection
4. HEPARIN
Mode of action: It inactivates thrombin hence fibrinogen not converted to fibrin
ADVANTAGE
- It’s an excellent natural anticoagulant
- It’s the best for osmotic fragility test
- Best for electrolyte determination
LIMITATIONS
- Expensive
- Causes coloration of blood smears
BLOOD SPECIMEN STORAGE
Changes occur when blood is allowed to stand invitro (outside the body) despite the
anticoagulant
To minimize the changes, the samples are stored at 4˚c
Lower temperatures than 4˚c enhance haemolysis
Higher temperatures above 8˚c enhance swelling of the cells
CHANGES BROUGHT ABOUT BY ANTICOAGULANTS
1. Red cells swell resulting in an increase in mean corpuscular volume (MCV)
2. Osmosis fragility (OF) and Thrombin time slowly increases, while Erythrocyte
sedimentation rate (ESR) decreases.
Leucocytes gradually autolyze (break with time due to the anticoagulants) but
hemoglobin remains unchanged.
All the above changes are slow.
Cold blood from a refrigerator should be first allowed to warm up to room
temperature and mixed properly before any test is performed or transfusion.

ANTICOAGULANTS EFFECTS ON CELL MORPHOLOGY

Blood having stood for 3 hours, especially oxalated blood, has marked changes,
especially leucocytes:
1. Some neutrophils may have
- Homogenously staining nuclei (difficult to differentiate the lobes)
- Separate nuclei bodies
- Coarse cytoplasmic marginal appearance
- Small vacuoles in the cytoplasm
2. Some mononuclear leucocytes develop changes as:
- Small vacuoles in the cytoplasm
- Nucleus undergoes irregular globulation which might end up in disintegration
3. Some lymphocytes
- Small vacuoles in the cytoplasm
- Nucleus budding resulting to nuclei with 2 or 3 lobes
- Some stain unusually homogenously
4. Red cells are little affected even after 6 hours at room temperature, though longer
periods lead to crenation and sphering but leucocytes in defibrinated blood are
hardly affected

BLOOD CELL COUNTS

Achieved in two ways: - By manual visual means
-Use of electronic hematological counters or hematological
analyzers
MANUAL COUNTS
APPARUTUS
Counting chamber (Hemocytometers)
Sucking tube with a mouth piece, cover slips, diluting pipettes, gauze
COUNTING CHAMBERS
They are of various types and include:
i. Improved Neubauer counting chamber
ii. Ordinary Neubauer counting chamber
iii. Burker counting chamber
iv. Fuchs- Roseuthal counting chamber
DILUTING FLUIDS
Fluids to be used as diluents must be isotonic and have a high specific gravity that
would prevent the cells from settling too fast.
Diluting fluids are grouped according to red blood cells, white blood cells and
platelets
1. RED CELLS COUNT
Fluids used are Toisons fluid, Hayems fluid and formal citrate.
HAYEM’S FLUID
Composed of:
Mercuric chloride - 0.5gms
Sodium chloride -1.0gms
Sodium sulphate -5gms
Distilled water -200gms
TOISON’S FLUID
Sodium chloride -1.0gms
Sodium sulphate -8.0gms
Glycerin -30mls
Distilled water -160mls
FORMAL CITRATE
Sodium citrate -3.0gms
Formaldehyde -1.0ml
Distilled water -100mls
2. WHITE BLOOD CELLS COUNTS
Diluting fluids include; Turks solution and Toison’s fluid colored with Gentian violet
(GV)
TURK’S SOLUTION
1% acetic acid tinged with GV
TOISON’S FLUID
The above (like for red blood cells), tinged with methyl orange
Add acetic acid or saponin to lyse the cells
3. PLATELETS
Baars fluid used as the diluting fluid and Ammonium oxalate
BAARS FLUID
Composed of;
Saponin -0.25gms
Sodium citrate -3.5gms
Brilliant creysl blue -0.1gms
Formation -1.0mls
Distilled water -200mls
Saponin lyses the red blood cells
Sodium citrate is an anticoagulant
Brilliant creysl blue stains the platelets
Formation fixes the platelets
Distilled water is used as a diluent
AMMONIUM OXALATE
1% of oxalate in 500mls of distilled water

RED BLOOD CELLS COUNTING METHOD

1. Blood is drawn into the red cell pipette up to the 0.5 mark
2. Draw in diluting fluid up to the 101 mark
3. Wipe the pipette with a piece of gauze
4. Close the tip of the pipette with the thumb, detach the sucking tube, place the
middle finger on the top and mix well by shaking for 2-3 minutes, making a 1:200
dilution.
5. Clean the counting chambers and cover glass with spirit. Place the chamber on a
flat surface and using a firm pressure slide the cover glass over the chamber until
Newton rings appear
6. Mix the suspension in the pipette, discard 1/3 of the mixture so as to expel the
diluting fluid in the stem
7. Charge the chamber by holding the pipette by hobbling it at an angle of 45˚and
slightly touching the tip against the edge of the coverslip
N.B; Do not overcharge the chamber as the cover glass will float and the
thickness of the chamber will vary hence giving wrong results
8. Place the chamber on the microscope stage and allow two minutes for the cells to
settle
9. Using a 4mm objective and ×10 eyepiece focus on the central square mm of the
counting chamber and count all the cells contained within 80 of a 400 small
squares
10. Count all cells touching the center line of the top and right-hand side and
disregard those touching lower and left-hand side of the center line
CALCULATION
Counted erythrocytes = Counted erythrocytes
Counted area× Height of chamber ×dilution 1̷ 5×1̷ 10×1̷ 200
Counted erythrocytes ×10,000 = Number of erythrocytes 1mm3blood
The area of the smallest square = 1̷ 80×5 = 1̷ 400mm3
The depth of the chamber = 1.0(1̷ 10) mm
The volume of one small square is 1̷̷ 400 ×1̷ 10×1 = 1̷ 400 mm3
Therefore, the volume occupied by the (N) cells counted is 1̷ 4000×80=1̷ 50mm3
Therefore 1̷ 50mm3 contain 1×N = N×50 = 50N cells
1̷ 50 1
Therefore, 1mm3of the undiluted blood will contain the 50N times the dilution factor,
which is 200
50 N ×200 = 10,000 N cells
Alternatively, N cells
1̷ 5×1̷ 10 × 1̷ 200
Where: 1̷ 50 = 1 of the small squares
1̷ 200 =Dilution factor
1̷ 10 =Depth of the chamber
Reference Values: -
Men 4.5-6.2 m/mm3
Women 3.9-5.6 m/mm3
Newborn 5.0-6.5 m/mm3
Children (1-10) 3.7-5.5 m/mm3

LEUCOCYTE COUNTS (WBC)

The anticoagulants used in white blood cells
APPARATUS

- Counting chamber
- White blood cells pipette
- Fluid

TECHNIQUE
1. Draw blood to the 0.5 mark of the stem of the white blood cells pipette and wipe
outside with a dry gauze
2. Draw the diluting fluid up to the 11thmark
3. Follow the rest procedure as the red blood cell count
4. Using the improved Neubauer chamber, count the cell in the 4 corner squares then
apply the game margin rule as for the red blood cells count.
5. Let N be equal to the number of cell counted is 4mm2volume in which N cells are
counted is
0.1 ×4=0.4mm3
N cells counted in 0.4mm3
X cells counted in 1mm3 in diluted blood
X= N×1 = 2.5 N
0.4
Since blood was diluted 1 in 20 therefore 1mm3of undiluted blood will contain
N×2.5×20= 50N cells
= Number of cells counted × Dilution
Number of large squares counted ×Height of chamber
REFERRENCE RANGES
Adults 4-11,000mm3(4.0-11.0×103/vol)
Infants 10-26,000mm3(10.0-26×103/vol)
Children (1-10) 6-17,000mm3(6-17×103/vol)
PLATELETS COUNT
BAAR’S FLUID
The principle is that saponin lyses red blood cells, Sodium citrate acts as
anticoagulant while formalin fixes the platelets and brilliant creysl blue stains the
platelets so that they appear as tiny refractive particles and the distilled water is a
solvent.
TECHNIQUE
Take anticoagulated blood to the 0.5 mark of the white blood cell diluting pipette
Take diluting fluid up to the 11thmark making 1:20 dilution
- Mix by shaking for 2-3 minutes
- Charge the chamber and wait for 15-20 minutes for the platelets to settle
- Place the charged chamber on a microscope stage and focus the central square
- Count as for the red blood cells
- Calculate same as for the red blood cells, except this time the dilution factor is
1:20 which will give 1000mm3
REFERRENCE RANGES
150,000-450,000 cells per mm3
N.B; When using blood from a finger prick, suck the diluent first up to the 0.5 mark,
then suck blood up to 1.0 mark and then suck diluent up to 11thmark, then proceed.
SOURCES OF ERROR IN COUNTING
 LOW COUNTS
1. Squeezing the site of puncture when filling the pipette
2. Insufficient blood being drawn into the pipette
3. Too much diluent being drawn into the pipette
4. Insufficient mixing
5. Using the first fluid expelled from the pipette
6. Insufficient filling of the counting chamber
7. Undue delay in performing the count, after filling the chamber
8. Using inaccurately calibrated pipettes
9. Faulty counting techniques
10. Saliva in the sucking tube which would increase the diluting factor
11. Errors in calculation
HIGH COUNTS
1. Using the first drop of blood expelled from the site of the puncture
2. Too much blood being drawn into the pipette
3. Insufficient diluent being drawn into the pipette
4. Insufficient mixing of the blood diluent suspension
5. Overfilling the counting chamber
6. Uneven distribution of the cells in the counting chamber
7. Inclusion of yeast and dust particles in the count
8. Inaccurately calibrated pipettes
9. Faulty counting techniques
10. Errors in calculation
RETICULOCYTE COUNT
When red blood cells near maturity they extrude their nuclei and the red blood cell at
this stage is called polychromatophilic cell. When the cells are stained with one of
the Romanowsky stains they are polymetachromatic cells and they give a bluish grey
color.
The presence of a number of these cells on a slide give a picture called
polychromasia.
The presence of these cells indicates an active erythropoiesis in the bone marrow
When a film shows a number of the immature cells, it suggests that reticulocyte count
or preparation is necessary to determine the percentage of retics present.
Retics are juvenile (young) red blood cells that are larger than mature red blood cells
They contain remnants of ribosomes and RNA which were present in large amounts
in the cytoplasm of the nucleated precursors from which they were derived
They exhibit a blue reticular when stained with supravital dyes. This is because
ribosomes contain a property of reacting with the dyes (New methylene blue, Brilliant
creysl blue, crystal violet and methyl violet), to form a blue precipitate of granules of
filaments.
The reaction only takes place in vitally stained and unfixed preparations.
N.B; The reticulum cannot be demonstrated with Romanowsky stain since they will
always stain polychromatic with it, because the fix the cells. The younger the
reticulocytes the more filamentous the reticulum. In older ones a small bluish dot
represents the reticulum.
New methylene blue is superior to Brilliant creysl blue as it stains the reticulum
material more deeply and uniformly than the latter which varies from sample to
sample in its staining ability.
TECHNIQUE/ METHOD
1. Place 2-3 drops of methylene blue or Brilliant creysl blue in a test tube
2. Add an equal volume of blood and mix
3. Incubate the mixture in a water bath at 37˚c for 15-20 minutes
4. Resuspend the mixture then make a thin smear on a clean glass slide and allow to
dry
5. Examine under oil immersion using ×100
N.B; Do not fix counterstain
6. Count at least 1000 red blood cells including reticulocytes and calculate the
percentage of retics present.
The reticular-filamentous material should stain deep blue and the non-reticulated
cells stained diffusely shades of pale-greenish blue.
Satisfactory counts may be made on blood which is not more than 24 hours old,
although the counts tend to fall as some of the retics may ripen in vitro
REFERENCE VALUES
Adults 0.2-2%
Infants 2-6%

SIGNIFICANCE OF RETICULOCYTE COUNTS

The retics count gives a fairly accurate reflection of the erythropoietic activity
That is the release of functional erythrocytes into the circulation. These release maybe
normal, increased or decreased.
INCREASED RETICULOCYTE COUNT CONDITIONS
1. Hereditary spherocytic anemia
2. Acquired auto-immune hemolytic anemia
3. Acute hemorrhagic anemia
4. Sickle cell anemia
5. Hemolytic disease of the new born
6. Treatment of iron deficiency, vitamin B12 and folic acid deficiency
7. Paroxysmal nocturnal hemoglobinuria (PNH) i.e. appearance of hemoglobin in
urine at night, diagnosed by HAM’s Test
DECREASED RETICULOCYTES COUNT CONDITIONS
1. Aplastic anemia
2. Pernicious anemia

HAEMOGLOBIN
Hemoglobin is a red conjugated protein, present in the red blood cells and is the one
responsible for the red color of the red blood cells.
The hemoglobin molecule is roughly spherical measuring 64×55×50A0 with a
molecular weight of 66,700
The large molecular weight of Hb makes it impossible for the Hb molecule to pass
through the glomerulus as something with molecular weight of 30,000 and below can
pass through the glomerulus.
Each red cell has 640 million molecules of hemoglobin

COMPOSITION OF HAEMOGLOBIN
Hemoglobin consists of two parts namely; Globin and Haem
Each Hb molecules consists of 4 groups of Haem attached to one globin
The Hemoglobin occupies 28% of a red cell mass, the rest being namely water and
some lipids

HAEM
It is a metal complex consisting of iron atom in the center of a porphyrin ring.
The iron is in ferrous state/form. Once in a ferric state, the Heam part gives the red
color to the hemoglobin
The porphyrin consists of 4 pyrole rings linked together by methane bridges to which
various side chains are attached.
The porphyrins form complexes with metal ions to produce various compounds e.g.
Hemoglobin which is an iron protophrin attached to the globin.
PORPHRIN MOLECULE
GLOBIN
It is a protenious substance composed of 4 polypeptide chains folded onto one another
to form globin.
The chains are attached to one another by electrostatic forces
These polypeptide chains are made of amino acids.
In a normal adult the globin part consists of two alpha chains and any other two
chains, Beta (β), Delta (δ), gamma, epsilon (δ).
Each of the Alpha chains consists of 141 amino acids
Each of the non-alpha chains consist of 146 amino acids
The globin part of Hb has a negative charge on the surface which facilitates its
combination with the Haem part
The Beta chains of the globin part could be substituted by other chains e.g. delta,
gamma and epsilon forming other normal Hb variants.
α chain synthesis is facilitated by chromosome number 16, while βchain is number 11
Total Amino acids in the globin are;
(2×141)+(2×146)=574
HAEMOGLOBIN SYNTHESIS
Hb synthesis is controlled by a number of genetic loci. The haem and globin are
synthesized separately and in different places but under normal circumstances, these
two biosynthetic processes appear to take place at the same time and similar rates.

HAEM SYNTHESIS
It takes place in the mitochondria (nucleus of the red blood cell).
The synthesis starts when glycine and succinyl Co A are condensed together in the
presence of
d-aminolevulinic acid.
Two of the d-aminolevulinic acid molecules are joined together in the presence of
glutathione and the enzyme d-aminolevulinic dehydrase to form porphobilinogen
(PBG) which is a monopyrole ring
4 monopyrole molecules of PBG condense to form uroporphyrinogen.
Uroporphyrinogen is then decarboxylated to form coproporphyrinogen which is
oxidized in the presence of another enzyme to protoporphyrin III.
An iron atom and protoporphyrin come together in the presence of Ferro chelatase
enzyme to form a Haem molecule.
 GLYSINE + SUCCINYL CO A

δ-ALA Synthetase

d-Aminolevulinic Acid

d-ALA dehydrase + glutathione

4 porphobilinogen

2 enzymes
Uroporphyrinogen

decarboxylatase
Coproporphyrinogen

Oxidation enzyme
Protoporphyrin III
Ferrochalatase enzyme

Fe++ HAEM

GLOBIN SYNTHESIS
The synthesis of globin takes place in the ribosomes.
Each structural chain acts on the DNA which transmits the basic genetic code from
the nucleus to the cytoplasm with the help of messenger RNA (mRNA)
The mRNA acts on the ribosomes after which the assembly of the amino acids starts.
Starting from the N-terminus the amino acids are added progressively towards the c-
terminus until the chain is complete.
After the completion of the chain they undergo a coil thus forming a helix.
4 of these helix chains are dimers (i.e. 2 alphas and 2 Beta) are brought together by
the molecular force to form the globin molecule.
Finally, 4 of the Haem molecules join up with a globin molecule to form a
hemoglobin molecule.
The process of transferring information from RNA to DNA is called transcription of
the genetic massage.
Ribosomes are composed of protein at a high molecular weight ribonucleic acid called
Ribosome RNA
NOTE; The alpha chains are coded in chromosome 16 while the non-alpha chains are
coded in chromosome 11. Transfer RNA (t-RNA) is soluble.

NORMAL HAEMOGLOBIN VARIANTS

There are 3 normal hemoglobin variants;
i. HAEMOGLOBIN A (HbA)
The Hemoglobin A (HbA) constitutes 95-97% of the total adult hemoglobin mass.
In the cord blood it is only in small amounts 15-40%
It consists of 2 alpha and 2 beta chains with the structural formula α2β2
Small amounts of HbA are detected in the foetus as early as the eighth week of life
During the first few months of post-natal life, HbA almost completely replaces HbF.
ii. HAEMOGLOBIN A2 (HbA2)
It comprises about 2-3% of total adult Hb mass.
Cord blood contains only traces of HbA2=0-1.8%
It comprises of 2 alpha and 2 delta with a structural formula of α2δ2
It occurs in some types of Thalassaemias and occasionally in megaloblastic anemia
and unstable hemoglobin disease.
HbA2 may be reduced in iron deficiency anemia and sideroblastic anemia.
iii. FOETAL HAEMOGLOBIN (HbF)
It is the predominant hemoglobin during the first few months and days after birth (3-6
months)
It is about 90-95% of total hemoglobin mass in infants
It is composed of 2 alpha and 2 gamma chains with a structural formula of α2Ϫ2
After the sixth month HbF decreases and a synthesis of beta chains start such that by
adult life, HbF is about 1-2%
HbF has a greater affinity for oxygen than HbA and HbA2 and as result of which it
carries a larger amount of oxygen
HbF may be elevated occasionally in cases of congenital and acquired aplastic anemia,
megaloblastic anemia, paroxysmal nocturnal hemoglobinuria, sideroblastic anemia
and in some forms of leukemia
It is occasionally raised in early pregnancy
HbF is measured by the alkaline denaturation test.

ABNORMAL HAEMOGLOBIN VARIANTS

They include;
1. HbS- Most important of the abnormal hemoglobin. It is associated with sickle
shaped cells in present in a red cell, distorting it.
2. HbC- Similar to HbS, but is not associated sickling.
3. HbE- Resembles HbC
4. HbD
HAEMOGLOBIN COMPOUNDS/ PIGMENTS
a. REDUCED HAEMOGLOBIN
Normally present in the blood accounting 5% of the total Hb in oxygenated blood
When blood is treated with dilute ammonium sulphide or some similar mild reducing
agents, the oxyhemoglobin present is reduced to hemoglobin

b. OXYHAEMOGLOBIN
Present in blood accounting for 60-95% of the total hemoglobin
When fresh blood is diluted with ammonia solution and shaken, the hemoglobin
liberated from the cells is converted into oxyhemoglobin by the oxygen present in the
solution
c. CARBOXYHAEMOGLOBIN
Formed upon or following chronic or acute exposure to carbon-monoxide
It causes death
Carbon monoxide combines irresistibly with hemoglobin
d. METHAEMOGLOBIN
Ferrous (Fe2+) ion becomes Ferric (Fe3+) and the resulting compound is called
methemoglobin or ferric hemoglobin
NOTE; Sulphanamide drugs lead to formation of methemoglobin
e. SULPHAEMOGLOBIN
Found in patients taking some oxidative drugs.
Sulphaemoglobin placed in a test tube, by adding a small quantity of ammonium
sulphide and potassium ferricyanide it produces a colored pigment which dilutes
blood
f. HAEMATIN
Formed from hemoglobin when the globin part of the molecule is split off and the
haem oxidized
Usually present in faeces after an alimentary tract hemorrhage
It can be produced in the test tube by treating blood with acid or alkali (Sahli or
alkaline haematin)
Occult blood is the invisible blood in stool
g. HAEMOCHROMOGEN
This is colorless compound
Haemochromogen is formed in the test tube by the reduction of alkaline haematin
with ammonium sulphide
h. METHAEMALBUMIN
Following rapid intravascular Hb in the plasma, is degraded and the haem portion
becomes attached to the plasma albumin resulting in the formation of
methaemalbumin.
HAEMOGLOBIN FUNCTIONS/ PROPERTIES
1. HAEM-HAEM INTERACTION
As haem is tetra, it carries more oxygen than myeloglobin which is a monomer
Thus, when one Haem group takes on oxygen, oxygen affinity in the remaining Haem
groups decreases.
When the second Haem is oxygenated, the affinity for the remaining is increased
Thus, oxygenation of one Haem group facilitates the oxygenation of the others.
All this facilitates the oxygen and carbon dioxide transport hence also having a
buffering effect.
Transportation of oxygen from the lungs to the tissues and also transportation of
CO2in the reverse direction.
O2+ Hb HbO2(oxyhaemoglobin)
As blood reaches the tissues where oxygen tension is low, the oxyhaemoglobin
dissociates and Oxygen diffuses in to the tissues
CO2+ H2O H2CO3 H++ HCO-3
Enzyme carbonic anhydrase drives this reaction. The reduced Hb (It has already given
up its oxygen to the tissues) has a very powerful buffering actions and neutralize the
H+ ions liberated in the above equation.
The resultant HCO-3 ions diffuse out of the red cell to unite with Sodium.
70% of the CO2 produced in tissues having been converted to HCO-3 is carried in
plasma as bicarbonate without upsetting the PH of the blood.
2. BOHR EFFECT
This is the effect of Hb becoming more acidic on oxygenation.
It comes about when reduced Hb is oxygenated producing a shift from a weaker to a
stronger acid hence H+ addition to the solution and when in reduced form, the reverse
happens.
The acidic PH facilitates rapid combination and release of oxygen to tissues even if
the oxygen concentration in the lungs is lower than that of the tissues.
3. 2,3-DPG
It is a red blood cell metabolite with oxygen affinity thus enhances subsequent
combination and release of oxygen to tissues irrespective of the oxygen combination
in tissues.
It’s because it reduces Hb easily.
HAEMOGLOBIN DISORDERS
1. Suppressive disorders
Suppression occurs in some anemias such as Thalassaemia where there is a
deficiency of precursor such as iron
2. Abnormalities
e.g. In the synthesis of porphyrins and porphyrinurias.
 The porphyria conditions are characterized by an abnormal excretion of
porphyrins.
Porphyrinurias are conditions in which excess of coproporphyrin III are excreted
probably due to block of haem synthesis between coproporphyrin III and
protoporphyrin IX.
HAEMOGLOBIN BREAKDOWN
When red blood cells are lysed, free haemoglobin is released
Haemoglobin breaks down and the ring structure of Haem is opened, iron is then split
off and the globin liberated.
The iron from the Haem goes to the Iron stores in the liver.
The protophorine is taken down to form biliverdin and finally indirect bilirubin
(alcohol soluble)
The indirect bilirubin combines with glucuronic transferase to form bilirubin
glucuronide
This is converted to urobilinogen and stercobilin which are passed out as waste
products.
In the intestine’s bilirubin glucuronide is converted to stercobilinogen and later
excreted in faeces as stercobilin.
Some of it is absorbed, reduced to urobilinogen and is excreted in urine as urobilin.
CHEMICAL CHARACTERISTICS OF HAEMOGLOBIN
1. Hb has electrophoretic mobility due to its amino acid content
2. It is soluble in water
3. It has a wavelength which is detected spectroscopically hence the Hb estimation it
absorbs a waveband at 540nm
4. It is denatured by acids, alkali and heat
5. It can be oxidized to methaemoglobin
6. It has sites for various substances e.g. oxygen, carbonmonoxide, carbon, lead,
cyanide
CHARACTERISTICS OF HB-F
1. Electrophoretically, it is slower than Hb-A
2. It is resistant to alkaline denaturation
3. It is twice resistant to acid elution as haemoglobin A
4. It is readily oxidized to methaemoglobin twice as fast as haemoglobin A
5. It has a high affinity for oxygen than Haemoglobin A and A2
ESTIMATION METHODS OF HAEMOGLOBIN
The aim of estimating haemoglobin concentration is to determine the oxygen carrying
capacity of blood. The results obtained will assist in determining disease which cause
a deficiency or excess of haemoglobin and in studying changes in hemoglobin
concentration before or after operation and transfusion.
The excess hemoglobin concentration will be found in such cases as polycythaemia
rubra vera.
Deficiency in amounts would be found in such cases as anemias (iron deficiency
anemia, pernicious anemia, aplastic anemia, megaloblastic anemia) chronic leukemia
(cancer of the white blood cells).
The determination of hemoglobin concentration can be achieved by use of various
methods. The following methods will be used:
1. Specific gravity method
2. Chemical methods
3. Spectrophotometric methods
4. Colorimetric methods
5. Electric methods

1. ACID HAEMATIN (SAHLI) METHOD

APPARATUS:
i Sahli tube
ii Standard tube
iii Stirring rod
iv 0.02ml pipette
v Sucking tube
vi Pasteur pipette
REAGENTS:0.1N Hydrochloric acid
 PRINCIPLE: Hemoglobin is converted to acid haematin by action of 0.1N
Hydrochloric acid which is thereafter mixed with the standard.
TECHNIQUE
a) Fill the sahli tube up to the 20thmark with 0.1NHcl
b) Add 0.02mls of blood using the Hb pipette
c) Mix well and let is stand for 5 minutes
d) By use of the Pasteur pipette, and either 0.1NHcl or distilled water drop-wise,
mixing between each addition with the stirring rod, until the color matches
that one of standard
e) Read the amount of solution in the sahli tube directly and report either in
grams or percentage

e.g. Given N% convert to grams

N ×14.6gm/dl
100
Gm%= gms/100mls
N.B:14.6gm/100ML is the standard concentration.
ADVANTAGES
a) It is cheap and easy to perform
b) It is portable
c) Little amount of blood is used
d) Can be used where there is no electricity
e) Standard is stable
DISADVANTAGES
a) It is the most inaccurate method
b) Since it depends on visual application, results obtained differ from person to
person because of difference in visual
c) Causes eye fatigue
d) You can only perform one test at a time

2. ALKALINE HAEMATIN METHOD

APPARATUS AND REAGENTS:
i Colorimeter
ii Hemoglobin standard (16.0%) Gibson and Harrison standard
iii 0.1m NaOH (sodium hydroxide)
iv 0.05ml pipette

PRINCIPLE: Hemoglobin is converted in alkaline haematin by the action of

Sodium Hydroxide.
The standard is commercially obtained and is 16gms% or
OD TEST ×concentration of standard
OD standard
TECHNIQUE
 a) Add 0.05ml of blood to 4.95mls of N/10 NaOH in a test tube
b) Mix well and boil for 4 minutes. At the same time, boil 5mls of the
standard solution.
c) Cool both tubes quickly in cold water and match the test against the
standard using a colorimeter with a green filter.
N.B: If the test gives too high a reading adds 5mls of distilled water to
both test and standard and read again, but remember to multiply the
result with a dilution factor.
Multiply by 2 then calculate the Hb contents in grams per 100mls of blood.
i.e. ODT × concentration of standard in gm/100ml
OD ST
Colorimeter test reading = 21
Colorimeter standard reading =28
Therefore, test Hb = 21 ×16= 12gms/100ml
28
As 14.6gms/100ml =100%
Hb% =x
X = 12×100
14.6
X = 82%
ADVANTAGES
a) More accurate than acid haematin method.
b) Use of colorimeter, therefore the difficulty of marching the colors does not
arise.
DISADVANTAGES
a) The standard is unstable
b) Can only be used where there is electricity
c) Cumbersome to perform because of the bleeding
d) Too much blood used

3. OXYHAEMOGLOBIN METHOD
APPARATUS AND REAGENTS:
i Colorimeter
ii 0.04% ammonia in distilled water
iii Standard Hb solution prepared by Thomson.
iv Test tube with a rubber band.
v 0.02Hb pipette
PRINCIPLE:
The hemoglobin is converted to oxyhemoglobin by dilution of ammoniated water.
Then the standard Hb solution is prepared as directed by the Thompson.
Then readings are taken by a colorimeter for both test and standard
TECHNIQUE
a) Add 0.02mls of blood to 4mls of ammoniated water in a test tube.
b) Mix well by inversion.
 c) Read OD of the standard solution, then OD of test in a colorimeter against
distilled water, then calculate amounts of hemoglobin in grams per 100mls
of blood.
NOTE: The method is only capable of estimating oxy-hemoglobin. The
other Hb pigments are not estimated.
ADVANTAGES
a) It is an accurate and quick method
b) No liquid standard is required for comparison

DISADVANTAGES
a) It produces an unstable pigment
b) It does not measure “inactive” hemoglobin
c) The personnel factor, although most of photoelectric colorimeters can be
adapted to carry out the oxyhemoglobin estimation.

4. CYANMETHAEMOGLOBIN METHD (DRABKIN’S TECHNIQUES)

APPARATUS AND REAGENTS
i Colorimeter
ii Standard solution of cyanmethaemoglobin
iii Drabkins solution
iv Test tubes
v 0.02ml pipette
PREPARATION OF DRABKINS SOLUTION
i Potassium cyanide-0.2gms
ii Potassium ferricyanide-0.12gms
iii Sodium bicarbonate-1gm
iv Distilled water, make the volume to one titre
PRINCIPLE
Blood is lysed by adding dilute drabkin solution (distilled water will do the lysing).
Ferricyanide then oxidizes the released hemoglobin iron from the ferrous state to
the ferric state to form methaemoglobin which then combined with potassium
cyanide to produce a stable hemoglobin pigment called cyanmethaemoglobin
which has an absorption peak at 540nm wavelength.
(Ferricyanide converts Hb to methaemoglobin. Cyanide converts methaemoglobin
to cyanmethaemoglobin)
Then read and record the OD’s against drabkins solution which is your bank. Then
multiply by the concentration and record in gms/100mls.
N.B: Commercial concentration is 18gms/100mls of distilled water. The
standard solution is commercially made.
TECHNIQUE
a) Add 0.02mls of blood to 4mls of darbkin solution in a test tube.
b) Mix well and allow the contents to stand for 10 minutes to allow full color
development to take place.
c) Take the readings in a colorimeter, using drabkins solution as the bank.
 d) Take the reading of the standard solution of cyanmethaemoglobin in the
same way.
e) Calculate concentrate of hemoglobin as follows:
OD of test × concentration of standard × dilution factor
OD of std
In gms/100ml of blood
A means Absorbance
T means Test
S means Standard
Divide by 1000 as standard solution is in mg/dl
(Hb) in gm/dl or 1000ml = AT × Conc S × dilution factor
As 1000
= AT × Conc S × 250
AS 1000
e.g. standard solution = 60mg/dl
Test absorbance reading = 30
Standard absorbance reading = 40
(Hb) = AT × Conc S × 250
As 1000

= 30 × 60 × 250
40 1000

= 45
4

= 11.3 gm/dl

PREPARATION OF STANDARD HB CURVE USING STANDARD

CYANMETHAEMOGLOBIN
The purpose of this curve is that in routine practice it could be consuming if standard
solutions were to be prepared with every batch of test.
Again, the standard solutions are saved i.e. to economize them by this method which
are otherwise expensive. Therefore, the Hb curve will help in taking the Hb
concentration from many different specimens of blood. The curve should be checked
or corrected at least every 15-20 days to check faulty of the instruments. When
preparing the curve, one may use either a commercially prepared hemolysate standard
or blood of very high Hb contents or packed cells or pooled cells.

PROCEDURE
1. Switch on the colorimeter and let it heat for about 30 minutes. While it is heating,
proceed as follows:
2. Take a commercially prepared standard hemolysate usually of 18gm/10ml of
blood.
3. Dilute the standard so as to have dilutions of 18,15,12,9,6 and 3 gm/100ml using
drabkins solution as diluent reagent.
Dilutions obtained using this formula: RC × RV
OC
4. Fill one clean cuvette with Drabkin’s reagent as blank and set the machine to zero
mark.
The filter is green (625nm) or wavelength scale at 540nm.
5. Fill another cuvette with the standard solutions to be read.
6. Starting with the lowest concentration dilution (3gms) place the solution in a clean
cuvette and wipe the outside with a clean gauze, then take the OD reading.
7. Do the same for all dilutions including the original standard solution i.e. the neat,
recording of the OD.
8. Make a chart and tabulate the results as follows:
CONC OF STANDARD (gm/dl) 18 15 12 9 6 3
DRABKIN’S SOLUTION (mls) 0 1 2 3 4 5
HEMOLYTASE SOLUTION 6 5 4 3 2 1
(ml)
TOTAL VOLUME 6 6 6 6 6 6
OD 40 34 27 20 12 5

9. Plot a linear curve of OD against Hb concentration in gms percent on the x-axis.

10. From this graph, prepare a table converting all the relevant OD readings to grams
per 100mls.
11. Take the given specimen of blood and make a dilution of 1 in 2oo that is 0.02mls
of blood in mls of Drabkin’s solution.
12. Take the OD in the same colorimeter and using the plotted curve, find the Hb
concentration in grams per 100mls.

ADVANTAGES OF CYANMETHAEMOGLOBIN METHOD

1. Cyanmethaemoglobin compound is very stable in solution, and can be kept up
to 6 years, therefore there is no hurrying in taking the readings after dilution.
2. All forms of the Hb are converted to cyanmethaemoglobin except
sulphamethaemoglobin, hence total Hb estimation.
3. Cyanmethaemoglobin standards are favorable.
4. More accurate and reliable.
5. Standard solutions can be prepared simply and less expensively from
hemolysates.

DISADVANTAGES
1. Employs apparatus that are not portable
2. Cannot be used in places without electricity
3. Potassium cyanide is used which is very poisonous
4. Color development takes a long time to be complete therefore causes delay in
reading.
NOTE:
a) Clouding cause false high results and can be due to: Hb S and Hb C (remedy is
dilute mixture 1:1 with distilled water. Read and multiply results with 2)
b) Exceptionally high WBC (remedy is centrifuge and use supernatant)
c) Abnormal globulins (remedy is add 0.1gm Potassium bicarbonate to
cyanmethaemoglobin reagent)
d) Lipemic blood (remedy is add 0.02ml patients plasma to 5ml Drabkin solution
and use as blank)
Normal Hb values:
Men 12.0-18.0 gm/dl
Women 11-5-16.5gm/dl
Infants 13.5-19.5 gm/dl