MINISTRY OF HEALTH OF UKRAINE

MINISTRY OF EDUCATION AND SCIENCE OF

UKRAINE
SUMY STATE UNIVERSITY

V.Yu. Garbuzova, L.А. Los, O.A. Obukhova

PHYSIOLOGY OF THE BLOOD

Educational book

Is recommended by the Central methodological study

with higher education the Ministry of Health of Ukraine

Sumy
Sumy State University
2010

3
UDK 612.1
BBK 67.9 (4УКР) я7
G72

Reviewers:
Shevchuk V.G. – doctor of medical sciences, professor;
Samokhvalov V.G. – doctor of medical sciences, professor;
Mischenko I.V. - doctor of medical sciences, professor

Is recommended by the Central methodological study with higher education

the Ministry of Health of Ukraine as educational book
for students of higher education IV level accreditation
(letter № 23-01-25/114 from 28.10.2009)

Garbuzova V.Yu.,
G72 Physiology of the blood: educational book / V.Yu. Garbuzova,
L.А. Los,
O.A. Obukhova – Sumy: Publisher SumSU. – 2010. – 165 p.
ISBN 978-966-657-272-4
This teaching aid described in the tutorial material for self-
preparing students to practical classes in physiology from the “Physiology
of the blood”. Each theme contains basic information theoretic question to
self, test questions and problems.
For students of institutions of the III - IV level of accreditation.

У навчальному посібнику викладений матеріал для

самостійної підготовки студентів до практичних занять з фізіології з
розділу “Фізіологія крові”. До кожної теми наведені основні
теоретичні відомості, питання до самопідготовки, тестові питання і
задачі.
Для студентів вищих медичних закладів III – IV рівнів
акредитації.

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 CONTENTS

INTRODUCTION…………………………………………… 4
Objectives……………………………………………………. 5
Chapter 1 Physical and Chemical Properties of the Blood….. 6
Chapter 2 Physiologies of Erythrocytes……………………… 34
Chapter 3 Blood groups…………………………………….... 62
Chapter 4 Protective functions of the blood. Leucocytes……. 85
Chapter 5 Haemostasis……………………………………...... 111
PRACTICAL WORKS……………………………………..... 138
Appendix I…………………………………………………… 163
Appendix II………………………………………………….. 165

INTRODUCTION

Blood is used to transport substances and together with

lymph and intercellular fluid belongs to the internal medium of
the organism. With the help of well regulated constant content
and properties of it, the internal medium provides comparably
independent existent of the organism in conditions of the
environment. Compounds of internal medium have common
and differential physical and chemical properties. They can

5
affect each other, and their state depends on activities of
various systems of the organism.
System of the blood is the variety of executive organs
(blood, which is circulated and stored; organs of blood
formation and blood degradation) and mechanisms of
regulation (nervous and humoral), activity of which is directed
on keeping the adequate changes of blood compounds to
provide adaptive reactions.
Blood participates in transport of substances, helps the
excretion of metabolic products, it provides protection from
antigens and non-protein factors, affects the regulation of
different functions of the organism.
Chapter “Physiology of the blood” is important in
preparing the medical doctor of any specialty, because the state
of internal medium describes all processes in organism, which
characterize homeostasis, homeokinesis and adaptive reactions
when changes in internal medium and co-operation of the
organism with the environment.
Teach chapter “Physiology of the blood” is necessary for
learning the next chapters of physiology and other subjects, for
example pathological physiology and all clinical medically-
specified subjects.

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Objectives:

 To know the definition of blood system, mechanisms of its

regulation based on analysis of homeostasis parameters:
blood volumes, acid-base balance, osmotic pressure,
quantity and quality content of blood plasma and formed
elements of the blood.
 To know physiological laws of functions of blood system:
respiratory, transport and protection.
 To know physiological laws of functions of keeping the
liquid consistency of the blood and development of
hemostasis when damage of blood vessels.
 To make conclusions about the state of physiological
functions of the organism, which perform with the help of
the blood system, based on quality and quantity showings:
hematocrit, number of erythrocytes, hemoglobin,
leucocytes, thrombocytes, leukogram, color index,
erythrocyte sedimentation rate (ESR), time of erythrocytes
coagulation, duration of bleeding.
 To analyze maturing changes of blood consistence,
function and mechanisms of regulation.
 To explain the physiological basis of examination methods
of functions of the blood system: quantity of formed blood
cells, hemoglobin, ESR, osmotic resistance of
erythrocytes, duration of bleeding, time of blood clotting,
definition of blood group in ABO and CDE.

7
CHAPTER 1 Physical and Chemical Properties of the
Blood

Blood is one of many functional systems of the organism.

Together with nervous system blood unties organs in entire
organism. At the same time they define the term – proper blood
system or physiological blood system (PBS).
The contents of PBS are:
1 Peripheral blood.
2 Organs or blood formation and blood degradation.
3 Mechanisms of nervous and humoral regulation of blood
content.

Functions of the Blood

Functions of the blood are important and various.

Practically all of them are associated with the circulation of the
blood in blood vessels. That is why the main function of the
blood is transport function. There are several types of it:
1 Respiratory function (transport of gases: O2 from the lungs
to tissues, CO2 from tissues to the lungs).
2 Trophic function (transport of nutrients from gastro-
intestinal tract and other organs to all the tissues of the
organism).
3 Excretory function (transport of metabolic products to the
excretory organs).
4 Regulatory function (transport of hormones and biologically
active substances from endocrine glands to aim-organs).
5 Protective function (transport of phagocytes and
immunoglobulins).
6 Thermoregulatory function (transport of heat from organs
that maintain warmness – liver and other internal organs to
the skin).

8
 Besides, pathogenic factors are transported with blood:
microorganisms, toxins, tumor cells. Transport of the last one
will lead to the development of metastasis of malignant tumor.
Except the transport function, blood plays an important
role in maintaining homeostatic features of the organism. That
is why the second important function of the blood is
homeostatic function. There are several types of it:
1 Maintenance of the constant chemical content and physical
properties of the blood (osmotic pressure, pH, temperature,
concentration of ions and other).
2 Maintenance of the constant volume of the circulating
blood.
3 Maintenance of the antigenic homeostasis.
The third, important function of the blood is creative
function. Macromolecules, which are transported with blood,
perform intercellular information transferring, which provides
regulation of intracellular processes of protein synthesis,
keeping the level of cell differentiation, renovation and
maintenance of tissue structure.

Content and Quantity of the Blood

Peripheral blood – is the blood, which circulates in

vessels and it is stored.
The volume of circulating blood (VCB) is 6-8% of body
weight of adult human or 70 – 75 ml/kg of the body weight
(approximately 4 – 6 liters).
VCB is an important physiological constant. VCB
depends on:
1 Age (VCB of the newborn is 10% of the body weight and
only in the period of pubescence it decreases to the level of
the adult).
2 Sex (men – 7-8%, women – 6-7% of the body weight).

9
3 Functional state of the organism (physically trained have
higher VCB, sportsmen can have up to 10%).
Normal VCB value is normovolemia, increased VCB –
hypervolemia, decreased VCB – hypovolemia.
Peripheral blood consists of plasma (55 – 60%) and
formed elements (40 – 45%) fig. 1.1.
Percentage volume of the formed elements is called
hematocrit. In normal state hematocrit value is HV almost
completely depends on the quantity of erythrocytes in the
blood, because its volume is nearly 99% of the volume of all
formed elements of the blood. Only in some forms of leucosis,
due to development of anemia and increase in quantity of
circulating leucocytes, the part of the last ones in hematocrit
value increases.

HV = (Red Cell Volume  Total blood Volume)  100

Hematocrit is defined by Wintrobe method. 2 ml of the

blood is put in special centrifugal glass, then add anticoagulant
to it and turn it for 10 minutes with 1000 turns per minute.
Blood cells, mass of which is higher than plasma will settle
down in the bottom. Due to plasma are lighter in weight than
erythrocytes, they will form thin white layer between
erythrocytes and plasma.

Plasma

Leucocytes

Erythrocytes
Thrombocytes

Figure. 1.1 – Compaund of the blood

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 Other method for determination of haemotocrite value is
microtechique: Blood sample is withdrawn in a heparinized
capillary glass tube of small size. Both ends are waxed. The
tube is put with other similar tubes in a special centrifuge and
rotated for few minutes. The tube is then removed after
separation of red cells from plasma and put on a special scale
to read the haematocrite value.
Hematocrit value depends on:
1) sex (men – 44% - 46%, women – 41% - 43%);
2) age (newborns have 20% higher, than women; children –
10% higher);
3) life conditions (due to adaptation to the mountain country,
hematocrit can increase);
4) HV is greater in venous than arterial blood (due to chloride
shift phenomenon) and in large, than in small vessels.
The increase of hematocrit can lead to increase of blood
stickness and that means to increase of the load on heart,
disorders of blood circulation.
Changes in haemotocrite value (HV)
1) HV is increased in:
a) polycythaemia: due to increased number of R. B. Cs as in
high altitude and in the newly born infants;
b) dehydration: due to decreased plasma volume as in severe
vomiting and diarrhea.
2) HV is decreased in:
a) anemias: due to decreased R. B. Cs. count;
b) Overhydration: as in renal diseases or after intravenous
infusion of large amounts of fluids (fig. 1.2).
Uses of haemotocrite value:
1) diagnosis of anaemias;
2) determination of blood volume and renal blood flow;
3) calculation of certain blood indices;
4) follow up of cases of shock.

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Functional importance of blood plasma components

Figure 1.2 – Haematocrit in the normal person and partients with anaemia
and polycythaemia

Main components of blood plasma are:

 Water (91%);
 Proteins (8%);
 Electrolytes (0,9%).

Importance of Water

1 Water medium, in which substances and blood cells are

dissolved.
2 Water defines VCB.

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3 It is needed to perform the exchange of the substances
between blood and intercellular fluid.
4 It affects reological properties of the blood (for example,
stickness).
5 Due to high thermal capacity it performs transport of heat.

Importance of Proteins

1 Transport function. There are special spots in the molecule

of protein, which are able to bind inorganic substances (for
example, ions, water) and organic substances (for example,
hormones, biologically active substances) and to transport
them. Binding of this substances to proteins provides:
a) Keeping small molecules in bloodstream when blood is
passing through kidneys.
b) Predicts its destructing by blood enzymes.
There are specific and non-specific transport proteins.
Not-specific – are able to bind different substances and
transport those (most of albumins transport hormones,
calcium). Specific – transport only one kind of substances. For
example, cerulloplasmin – ions of cuprum (Cu), transferrin –
ions of iron (Fe), haptoglobulin – bilirubin.
2 Trophic function. Proteins are the source of amino acids,
which with peripheral tissues, are used for the formation of
proper, specified for the organ proteins. Proteins are the source
of energy. When breaking down 1 g of protein in the organism
4,1 kkal.
Trophic function of proteins is used clinically when
disorder of natural way of nourishment, in parenteral
nourishment, when protein suspensions are injected in
bloodstream.
3 Enzymatic function. There are a lot of protein-enzymes into
the plasma. There are secretory and indicatory (cellular)
enzymes. Secretory enzymes are synthesized in liver and are

13
excreted in the blood plasma, where they perform their
function. Typical representatives of this group are protein-
enzymes of blood clotting. Indicatory enzymes are entering the
blood from other organs. Their activity is not high. In
conditions of pathological states, enzymes are taken from cells
into the blood and its activity increases, which indicate the
level of lesion. That’s why quantitative definition of blood
enzymes is one of the available laboratory methods of
diagnostics. For example, activity of AlAt
(alaninaminotransferase) increases during liver sickness.
Activity of AsAt (aspartateaminotransferase) increases up to 20
times during myocardial infarction. Activity of
lactatedehydrogenase increases during myocardial infarction,
hepatitis, myopathy, tumors, and leucosis.
4 Participating hemostasis. Proteins comprise biochemical
systems of blood plasma, which provides hemostasis, namely:
 Blood clotting system;
 Anticoagulatory system;
 Fibrinolytic system;
 Kallikrein-kinine system.
5 Participating in maintaining pH of the blood. Proteins form
protein buffer. In acidic medium, they work as bases, binding
acids; in base, they react like acids, binding bases. This
property of the proteins is called amphoteric. Mostly buffer
properties belong to carboxyl groups and amino groups.
Plasma proteins are responsible for 15% of the buffering
capacity of the blood and carriage of CO2.
- Protein – NH2 +CO2  NHCOOC (Carbamino protein);
- Proteinic acid: Na proteinate buffer:
a) Na proteinate +H2CO 3  NaHCO3 + Proteinic acid.
b) Na proteinate + lactic acid  Na lactate + Proteinic acid.
Lactic acid (strong acid) is converted to proteinic acid
(weak acid).

14
6 Maintenance of the reological properties of the blood,
namely its stickness. When increase in protein quantity,
stickness increases, when decrease in protein quantity,
stickness decreases.
7 Proteins are the source of biologically active substances.
For example, kinins and angiotensin.
8 Protective function. Proteins participate in non-specific and
specific protection of the organism. Non-specific protection is
represented by complement system proteins, interferons,
orosomucoid and viruses’ inhibitors. Specific – by antibodies:
congenital (agglutinins) and acquired.
9 Making creative connections. Proteins participate
transferring of the information, which affects genetic apparatus
of cells, provides growth, development and differentiation of
tissues. For example, proteins are growth factor of nervous
tissue, erythropoietin etc.
10 Capillary Permeability. Plasma proteins close the pores in
the cement substance (between the endothelial cells) of the
capillary wall. Hypoproteinaemia increases the capillary
permeability.
11 Creation of oncotic. (colloid-osmotic) pressure
Ponc= 25 – 30 mmHg. 80% of oncotic pressure is made by
albumins (molecule of albumin has small size and in volume of
plasma, its quantity is the highest).
Role of oncotic pressure in redistribution of water in the
organism (fig. 1.3):

15
 C a p i l l a r y

Arterial part Venous part

Рhbp = 32,5 mmHg Рhbp = 17,5 mmHg

Рobp = 25 mmHg Рobp = 25 mmHg

Filtration Reabsorbtion

I n t e r c e l l u l a r l i q u i d

Рhp = 3 mmHg Рhp = 3 mmHg

Рop = 4,5 mmHg Рop = 4,5 mmHg

Figure 1.3 – Redistribution of water in the capillaries

Wall of capillaries is permeable to small molecules and water.

That is why osmotic pressure in blood plasma and in intercellular
liquid is almost equal. Big molecules, first of all protein molecules,
can not pass through the capillary wall. That’s why there is gradient
of protein concentration (oncotic pressure gradient - P onc) between
plasma and intercellular liquid. Ponc inside capillary is higher than in
intercellular liquid. Hydrostatic pressure – pressure of the liquid on
the capillary wall (from one side blood is making pressure on
capillary wall, from other side – intercellular liquid), it also plays

16
important role in redistribution of the water. Hydrostatic pressure of
the blood is higher, than hydrostatic pressure of intercellular liquid.
Water exchange occurs in two ways:
1) filtration (transition of the water from the capillary to the
tissue);
2) reabsorbtion (transition of the water from the tissue to the
capillary).
The direction of the water movement is defined by the
filtrative pressure (Pf).
Pf = (Phbp + Pop) – (Php + Pobp)
Phbp – hydrostatic pressure of the blood;
Pop – oncotic pressure of the intercellular liquid;
Php – hydrostatic pressure of the intercellular liquid;
Pobp – oncotic pressure of the blood.
If Pf >0 – filtration occurs.
If Pf <0 – reabsorbtion occurs.
Increasing of the Phbp and Pop leads to filtration, increasing
of the Php and Pobp leads to reabsorbtion.
In the arterial end of the capillary:
Pf = (32,5 + 4,5) – (25 + 3) = 9 mmHg – filtration occurs, water
is transited to the tissue.
As the blood passes in the capillary, in the result of
transition of the water to the tissue, hydrostatic pressure
decreases. In the middle of the capillary Pf = 0 and water
transition stops.
In the venous end of the capillary:
Pf = (17,5 + 4,5) – (25 + 3) = - 6 mmHg – reabsorbtion occurs,
water passes into the capillary.
At the beginning of the capillary approximately 0,5% of
blood plasma passes to the tissues. P f in the arterial part of the
capillary (Pf = 9 mmHg) is higher than in the venous part (P f =
- 6 mmHg), that is why the bloodstream returns not the whole
100% of the liquid, but nearly 90%. 10% are excreted through
the lymphatic vessels.

17
 This pressure values may vary in different organs and it
depends on organ activity. Described mechanism of filtration –
reabsorbtion is called Starling’s mechanism.
Changes in any of the parameters may cause disorder in
filtration and reabsorbtion correlation.
For example, the decrease in protein concentration in
blood plasma will lead to the decrease of the reabsorbtion,
delay of the water in intercellular medium and development of
the intercellular oedema. This can happen during starvation
(cachexy oedemas); when pathological processes in kidneys, in
the consequence of which proteinuria can occur and loss of
proteins (nephrotic oedemas); when disorder in albumin
synthesis by liver (hepatic oedemas); when allergic and
inflammatory processes, when there is the increase of vessel
wall permeability and other plasma proteins are leaving to the
intercellular space (membranogenic oedemas) and other.

Plasma protein consistency

Plasma proteins can be divided into several fractions:

1 Albumins (35-50 g/l). They are mostly small proteins; their
molecular mass is not more than 70000 Da, Synth esized by the
liver only. They are circulating in the bloodstream for a long
time: the period of halfexcretion is 10-15 days. The main
functions of albumins are transport and trophic. Due to high
hydrophilic, small sizes and high concentration in blood
plasma albumins play important role in the creation of oncotic
pressure. Decrease in albumin concentration to 30 g/l and
lower can lead to the decrease of oncotic pressure and the
development of oedemas.
2 Globulins (20-40 g/l): α1, α2, β, γ. Molecular mass is 44000-
130000 Da, synthesized by the liver and reticuloendothelial
cells as well as plasma cells present in the lymph nodes, spleen,
bone marrow and liver. The term of circulation of globulins is

18
less than the one of albumins: the period of halfexcretion is less
than 5 days. The main functions of globulins are transport and
protective.
3 Fibrinogen (2-4 g/l). It is the largest protein of blood
plasma. Synth esized by the liver only. Fibrinogen plays a role
in processes of blood clotting and thromb formation.
The correlation between albumins and globulins is called
albumin-globulin coefficient or albumin – globulin ratio.
Normally it is 1,5 - 2,3.
Clinical importance of Albumin – Globulin ratio (A/g
ratio):
1 A/g ratio is decreased in:
a) liver diseases (e.g.hepatitis) due to decreased albumin
formation;
b) renal diseases (e.g. nephrosis) due to albumin loss in
urine;
c) infections and allergy due to increased synthesis of -
globulins.
2 A/g is increased in:
a) hypogammaglobulinaemia;
b) acquired immunodeficiency syndrome (AIDS) due to
decrease - globulins.

Importance of Electrolytes

The main function of blood plasma electrolytes is the

creation of osmotic pressure. Posm = 7,5 atm (0,3 osmole, 745
kPa, 5600 mmHg). The osmotic pressure is defined by the
quantity of solved particles, not by the size of them. 96% of the
osmotic pressure is made by Na+ and Cl- ions, because the
molecular mass of NaCl is small.
Solutions, the osmotic pressure of which is the same as
blood plasma has, are called isotonic. For example, 0,9%
solution of NaCl, Ringer’s solution, Ringer-Lock’s solution,

19
Tirode, 5% glucose solution, haemodesis. Solutions with the
osmotic pressure of which is higher than the plasma is called
hypertonic, if it is lower it is called hypotonic.
In hypertonic solution water is leaving the cells, cells
firm, their normal turgor is disbalanced. It is called
plasmolysis. In clinics it is used for counting the erythrocytes:
blood is diluted by 4% solution of NaCl, erythrocytes are
firmed and they are easy to count.
In hypotonic solution water enters cells, cells swell,
oedema of the cells occurs and destruction of the cells, which is
called haemolysis. In both cases cells metabolism disorder
occurs or even can lead to the death of the cell.

Plasmolysis Haemolysis

Figure 1.4 – Plasmolysis and haemolysis in erithrosyts

Demands to blood substitutes

1 Isotonicity. Osmotic pressure of blood substitutes should be
equal to Posm of plasma. 0,9% solution of NaCl is the
simplest blood substitute.
2 Balanced content of inorganic salts.
3 Isooncoticity. Big molecules are slowly excreted from the
bloodstream, it helps extended refresh of volume of
circulating blood (VCB). This is reopolyglukin, haemodesis,
polydesis.
4 pH should be equal to the pH of blood plasma.
5 Sterility.
6 It should be non toxic.

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 Classification of blood substitutes
Group 1 – hemodynamical:
Low molecular dextranes – reopolyglukin;
Medium molecular dextranes – polyglukin;
Gelatin substances – gelatinol.
Group 2 – disintoxicational:
Low molecular polyvinylpyrolidon – heamodesis;
Low molecular polyvinyl alcohol – polydesis.
Group 3 – preparations for parenteral nourishment:
Protein hydrolysates – casein hydrolysate,
Aminopeptide, aminokrovin, aminosol, hydrolysin;
Solutions of amino acids – polyamine, maryamin,
freeamin etc;
Fatty emulsions – intralipid, lipofundin;
Sugars and polybasic alcohols – glucose, sorbitol,
fructose.
Group 4 – regulators of fluid-electrolyte and acid-base balance:
saline solutions – isotonic solution of sodium chloride,
Ringer’s solution, lactosol, solution of sodium
hydrocarbonate, trisamin solution, etc.
Functional system, which provides constancy of the
osmotic pressure.
Osmotic pressure is an important physiological constant.
Any deviation of osmotic pressure from normal will lead to
redistribution of water between cell and intercellular medium.
For maintenance of the osmotic pressure on the same
level in the organism there is a functional system, which
consists of external and internal part. In basis of external part
there are behavioral responses, which are responsible for
normalizing of osmotic pressure. If there is increase in osmotic
pressure, human feels thirsty and drinks water. If there is
decrease in osmotic pressure, human will feel to eat something
salty. In basis of internal part there are local mechanisms of
reflexes. Local mechanisms are the processes, which occur in

21
blood itself. If decrease of osmotic pressure, excess water is
connecting to the low molecular proteins, formed blood cells
and Posm increases. If increase of osmotic pressure, excess
electrolytes salts is absorbed by formed blood cells and
transported to organs (K+, Ca2+ - to muscles, Ca2+, PO43- - to
bones, Na+, Fe2+ - to the liver, Na+, Cu2+ - to the spleen, Na+,
Ca2+, Zn2+ - to the pancreas) and Posm decreases.
These mechanisms are working during few hours and if
Posm will not go to normal, local mechanisms of reflexes will
start working.
There are 3 reflexes for the maintenance of Posm:
1) osmoregulative;
2) volumeregulative;
3) Na-uretive.

Osmoregulative Reflex

When increase of osmotic pressure osmoreceptors are

irritated (peripheral – in vessels, heart, liver and central – in
hypothalamus).
From osmoreceptors impulses are passing to supraoptical
and paraventricular nuclei of hypothalamus, where ADH
(antidiuretic hormone is formed). It is transported to the
neurohypophysis through axons and it is released into the
blood. It is the main hormone, which retain water in the
organism.
Its targets are distal tubules and collecting tubules of the
nephron. In distal tubules ADH interacts with α-receptors on
basolateral cell membrane, excretion of hyaluronidase from
cells activates, which breaks down hyaluronic acid from
intercellular space, in result the permeability of epithelium of
tubules for water increases. In tubules, ADH interacts with V 2-
receptors, adenylcyclase system activates, formation of cAMP
increases, then cAMP diffuse in apical membrane, which

22
activates membrane permeability to water, ADH interacts with
V1 in blood vessels. It leads to formation of inositol-tri-
phosphate (ITP) and diacylglycerol (DAG), decrease of cAMP
amount and constricting of vessels. Due to this effect, there is
another name of hormone – vasopressin.
Decrease of osmotic pressure is caused by decrease of the
sodium level in blood plasma. Hyponatriemia stimulates
secretion of renin. Renin activates formation of angiotensin II,
which stimulates secretion of aldosterone by adrenal glands
cortex. The targets of aldosterone are distal tubules of nephron,
where it increases reabsorbtion of sodium ions, restores normal
sodium concentration in plasma. In result, osmotic pressure
increases.
Volumeregulating Reflex

This reflex starts working when VCB decreases by 7 –

15% (this change goes with increase of P osm). Excitation of
volumereceptors in vessels, internal organs and cavities occur.
From volumereceptors, impulses are passing to hypothalamus
to supraoptical and paraventricular nuclei. Here ADH is
formed, which increases reabsorbtion of water in kidneys and
causes increase of VCB and decrease of Posm.

Na-uretive Reflex
This reflex works when VCB increases. In result volume
of blood that is passing to the heart, increases. Atrium walls
overexpand. In the consequence of this myoendocrine cells of
both atria release Na-triuretic hormone (atriopeptin). Target of
this hormone is distal tubules of kidneys, where it decreases
reabsorbtion of sodium, and, as a consequence, Na-uresis,
diuresis increase, VCB decreases, Posm increases.

Regulation of Secretion

23
 Four important factors are involved in the regulation of
secretion of aldosterone. The stimulatory agents for aldosterone
secretion are:
1) increase in potassium ion concentration in the extracellular
fluid;
2) decrease in sodium ion concentration in the extracellular
fluid;
3) decrease in extracellular fluid volume;
4) adrenocorticotropic hormone.

Mechanism

Increase in the concentration of potassium ions is the

most effective stimulant for aldosterone secretion. It acts
directly on the zona gromerulosa and increases the secretion of
aldosterone. Decrease in sodium ion concentration and
extracellular fluid volume stimulates the juxtaglomerular
apparatus in the kidney to secrete renin. Renin acts on
angiotensinogen in the plasma and converts it into angiotensin
I, which is converted into angiotensin II. Angiotensin II acts on
the zona gromerulosa to secrete more aldosterone. Aldosterone
in turn, increases the retention of sodium and water. This
causes an increase in the sodium ion concentration and volume
of extracellular fluid.
Now, the increased sodium ion concentration and the
extracellular fluid volume inhibit the juxtaglomerular apparatus
and stop the release of renin. So, angiotensin II is not formed
and release of aldosterone from adrenal cortex is stopped.
Adrenocorticotropic hormone mostly stimulates the
secretion of glucocorticoids more than aldosterone. Still, it has
mild effect in stimulating aldosterone secretion.

Physical and Chemical Properties of the Blood

24
1 Osmotic pressure. Posm = 7,5 atm
2 Density. Defined by presence of soluble substances.
pof plasma = 1,025 – 1,034 g/cm3
pof blood = 1,050 – 1,060 g/cm3
3 Stickness. Stickness – internal friction, which is made by
friction of formed elements between each other and with
vessel wall. Stickness resistance to bloodstream. Stickness
of the liquid is defined comparatively to water, stickness of
which is 1.
Stickness of plasma = 1,7 - 2,2
Stickness of blood = 5.

Factors, which affect stickness:

1) hematocrit (the higher number of erythrocytes - the

higher the stickness);
2) the amount of proteins (the higher amount of proteins -
the higher the stickiness).
4 Active reaction of the blood (pH).
pH of the blood – is reversed logarithm of the
concentration of H+ ions. pH is determined by correlation of H+
ions and OH- in blood.
pH of the arterial blood = 7,4
pH of the venous blood = 7,36
Decrease of pH is called acidosis. Increase of pH is
called alkalosis.
Durable change of pH even on 0,1 - 0,2 may be lethal.
The borders of pH changes, which are compatible with life, are
7,0 – 7,8. But these changes should not be durable, because the
disturbance of pH level can lead to death.

Mechanisms which provide the constant pH level

25
 Since pH of the blood is one of the most important
homeostatic factors, its maintenance on the constant level is
provided by many organs and systems of the organism.
The first “mean of protection” for the constant pH are
buffer systems of blood. Every buffer system consists of two
substances – weak acid and strong base. In process of
metabolism formation of acid products is faster than basic
products that are why the danger of acidosis in organism is
higher. That is why in buffer pair acid-base, base capacity is
higher and buffer systems are more resistible for the influence
of acids. So, for the change of pH to the base side we should
put 40 - 70 times more of NaOH, than to water, and to change
pH to the acid side 300 - 350 times more of HCl.
There are 4 buffer systems of blood:
1) hydrocarbonate;
2) phosphate;
3) hemoglobin;
4) protein.

Hydrocarbonate buffer system consists of carbonic

acid – H2CO3 and sodium hydrocarbonate – NaHCO3 in
correlation 1:20. Principle of its functioning lies in: when acid
is entering the blood (for example lactic acid, C3H6O3), which
is stronger than carbonic acid, base reserve provides exchange
of ions with formation of carbonic acid, which dissociates on
CO2 and H2O.

СН3– СН–СООН + NaHCO3 CH3 – CH – COONa + Н2CO3

OH ОН CO2 H2O
Lactic acid

26
 This process is especially active in lungs, where CO2 is
immediately loosed from the organism, which provides
maintenance of pH on the constant level and prevents acidosis.
In case basic products enter the blood, the acid reservoir
provides exchange of ions with the formation of bicarbonate
and water:
R+OH¯ + H+HCO3¯ RHCO3 + H2 O

RHCO3 is used to refill the buffer or released through

kidneys. Usage of НСО3¯ leads to the leakage of CO2 and
releasing of it through the lungs decreases.
So, hydrocarbonate buffer is the most mobile, powerful
enough (its capacity is 13%), it is closely related with
respiratory system.
Phosphate buffer system consists of acid sodium salt of
phophatic acid (NaH2PO4) and basic (Na2HPO4) in correlation
1:4. It is functioning by the same principle, like hydrocarbonate
buffer. Due to low quantity of phosphates in blood, its capacity
is low (5% of total capacity).
Hemoglobin buffer is represented by deoxidated
hemoglobin (HHb) and potassium salt of oxidated hemoglobin
(KHbO2) (75% of total capacity)
In capillaries of the tissues due to accumulation of acid
metabolites there is danger of blood acidosis and hemoglobin is
behaving like a basic substance:

KHbO2 → O2 + КHb (reaction of deoxygenation)

Hemoglobin, separated from oxygen has higher ability to

bind H+ protons.
KHb + HR¯ → HHb + KR¯
HHb + CO2 → HHbCO2
ННbCО2 is transported to the lungs
HHbCO2 → CO2 + HHb
27
 In lungs due to release of CO 2 there is danger of blood
alkalosis and hemoglobin behaves like an acid.
HHb:
1) the source of protons: HHb → H+ + Hb¯;
2) supports formation of H2CO3, which also dissociates with
formation of protons:
HHb + KHCO3 KHb + H2CO3

Н+ HCO3¯
Protein buffer system is represented by proteins, which
in acid medium behaves like bases, binding acids, in basic
medium they react like acids, binding bases. Amphotericity of
proteins is determined by amino acids, especially by carboxyl
groups and amino-groups.

COOH - the source of Н +

R
NH2 - binds Н+
Buffer systems are not only in blood, but also in tissues,
where they keep pH on the constant level. The main buffer
systems of the tissues are protein and phosphate buffer.
Buffer systems do not only reduce the pH shift, but they
also predict total changes in pH level. That’s why for the
effective maintenance of acid-base balance, other organs and
systems are involved. For the fast compensation of pH lungs
are involved due to its ability to regulate the amount of
excreted CO2. The compensatory reactions of kidneys in form
of decrease of hydrocarbonate reabsorbtion, acidogenesis and
amoniogenesis, develop after 6-12 hours, or even days. Other
organs also work to maintain of constant pH level. So, sweat-
glands are able to excrete some products of metabolism (lactic

28
acid), liver uses lactic acid of blood for glycogen biosynthesis,
heart uses lactic acid like a substrate for oxidation.

Features of acid-base state of the blood

1 pH = 7,36 – 7,4.
2 Partial pressure of CO2 of the arterial blood (pCO2) = 35 –
45 mmHg.
3 Actual bicarbonate of the blood (basic reserve of the blood)
– the true concentration of bicarbonate ion HCO3¯ in
practical state of the arterial blood in the bloodstream.
AB = 22 – 25 mmol/l
4 Standard blood bicarbonate (SB) – the concentration of the
bicarbonate ion in full saturation of the hemoglobin with
oxygen (shows the shift of pH, which is not associated with
breathing). Normally SB = AB.
5 Buffer bases (BB) – the total amount of concentrations
of all ions in the blood, which have buffer properties in full
saturation of the blood with oxygen.
BB = 40 – 48 mmol/l.
The special feature of it is that its value does not depend
on changes of pCO2 that allows estimating the condition of
acid-base balance of the organism independently from
respiratory and not respiratory (metabolic) functions.
6 Buffer excess (BE) – the difference between the amount of
buffer bases in patients organism and normal values.
HBO = -2,5 + 2,5

Age features of physical and

chemical properties of the blood

Newborns and babies of the first year have different

features of the blood than adults. So, newborns have higher
density and stickiness of the blood, which is determined by
higher erythrocytes concentration. Till the end of the first
29
month of life, these features decrease and approach to the one,
adults have, or they become lower.
Placental blood circulation and labor complicate
interchange of gases. That is why children have acidosis before
birth (pH = 7,13 – 7,23). During the first hours (or days) after
birth, acidosis gradually disappears.
The concentration of plasma proteins in newborn
organism is lower (50 – 56 g/l). It will reach the level of adult
in age of 3 – 4 years. The high concentration of γ-globulins is
specific for the newborn, which the newborn gets from the
mother. Till the end of third month its content decreases, but in
future, with the help of its own antibodies formation, it will
gradually increase. The concentration of α and γ-globulins
reaches the adult level till the end of the first year of life.
With age, most of the physical and chemical properties of
the blood (pH, osmotic pressure, sodium and potassium
concentration, viscosity), stay on the same level. Other features
can change. So, ECR increases, osmotic resistance of
erythrocytes, hematocrit, ablolute and relative albumins
concentration decrease.

Questions for self-control:

1 Functions of the blood.

2 The volume of circulating blood (VCB). Factors, which de
termine VCB.
3 The composition of peripheral blood.
4 Hematocrit. Factors, which determine hematocrit. Methods of
hematocrit definition.
5 The importance of water.
6 The composition and functions of plasma proteins.

30
7 The role of oncotic pressure in redistribution of water in
organism.
8 The importance of electrolytes.
9 The definition of isotonic, hypotonic and hypertonic
solutions.
10 Requirements to blood substitutes.
11 The definition of plasmolysis and heamolysis.
12 Osmotic blood pressure. Functional system, which provides
the consistency of osmotic pressure.
13 Physical and chemical blood properties.
14 Active reaction of the blood. Mechanisms of maintenance of
constant pH level.
15 Principles of buffer systems functioning.
16 Features of acid-base balance of the blood.

Tests for self control:

1 The part of blood volume, which is occupied by erythrocytes:

a) volume index;
b) stickiness;
c) erythrocyte sedimentation rate (ESR);
d) color index;
e) hematocrit;
f) no correct answer.

2 Cations of blood plasma are:

a) sodium;
b) chlorine;
c) bicarbonate;
d) potassium;
e) calcium;
f) phosphate;
g) magnesium;

31
 h) sulphate.

3 Name the main function of blood plasma electrolytes:

a) formation of hydrodynamic pressure;
b) formation of hydrostatic pressure;
c) formation of oncotic pressure;
d) formation of osmotic pressure;
e) formation of blood stickiness;
f) protein transport;
g) no correct answer.

4 The main role in the formation of oncotic pressure is:

a) electrolytes;
b) albumins;
c) globulins;
d) erythrocytes;
e) leucocytes;
f) no correct answer.

5 What are the functions of plasma proteins:

a) participating hemostasis;
b) protecting function;
c) transport function;
d) no correct answer?

6 Name the cases when hematocrit decreases:

a) the increase of the erythrocytes amount in blood;
b) the decrease of the erythrocytes amount in blood;
c) the decrease of the blood volume;
d) the increase of the blood volume;
e) no correct answer.

7 The amount of what cells determines blood stickiness:

32
 a) monocytes;
b) lymphocytes;
c) thrombocytes;
d) eosinophils;
e) basophiles;
f) erythrocytes;
g) neutrophils;
h) no correct answer?

8 Name the components of hydrocarbonate buffer system:

a) sodium hydrocarbonate;
b) potassium hydrocarbonate;
c) magnesium hydrocarbonate;
d) carbonic acid;
e) hydrochloric acid;
f) no correct answer.

9 In normal state pH of the arterial blood is:

a) 7,32;
b) 7,36;
c) 7,40;
d) 7,44;
e) no correct answer.

10 Name the changes, when the filtration rate in capillaries will

decrease:
a) the increase of oncotic pressure of the blood in
capillaries;
b) the decrease of oncotic pressure of the blood in
capillaries;
c) the increase of oncotic pressure in the intercellular
medium;
d) the decrease of oncotic pressure in the intercellular
medium;

33
 e) the increase of the hydrostatic pressure of the blood in
capillaries;
f) the decrease of the hydrostatic pressure of the blood in
capillaries;
g) the increase of the hydrostatic pressure in the
intercellular medium;
h) the decrease of the hydrostatic pressure in the
intercellular medium.

11 What is the value of the osmotic pressure in the normal

state:
a) 7,5 atm;
b) 7,6 mmHg;
c) 56 atm;
d) 74 mmHg;
e) no correct answer?

12 When pH of the blood is 7,32, it means:

a) normal state;
b) acidosis;
c) alkalosis;
d) hypovolemia;
e) hypervolemia;
f) no correct answer?

13 What happens to the cell in hypertonic solution:

a) oedema;
b) haemolysis;
c) plasmolysis;
d) immediate death;
e) no correct answer?

14 What is the normal quantity of the blood:

a) 1/5 of the body mass;

34
 b) 10 – 15% of the body mass;
c) 6 – 8% of the body mass;
d) 4 – 5% of the body mass;
e) no correct answer?
15 What are the main buffer systems of tissues:
a) hemoglobin buffer;
b) hydrocarbonate buffer;
c) phosphate buffer;
d) protein buffer;
e) no correct answer?

35
CHAPTER 2 Physiologies of Erythrocytes

Definition of erythron

Erythron is the total mass of erythrocytes in the organism

(the one circulating, the one that are stored, the one in organs
of formation and degradation), and also the mechanisms of
regulation of their amount.

Functions of erythrocytes
1 Respiratory function. Transport of the oxygen – is the
main function of erythrocytes, because this function in
human organism is performed only by them.
2 Transport function. Transport of CO2, proteins, hormones.
3 Buffer function. Maintaining of pH level in blood with the
help of hemoglobin buffer system.
4 Maintaining the rheological properties of blood, namely
stickiness (increasing the amount of the erythrocytes leads
to increasing the stickiness, when decreasing – decreasing
the stickiness).
5 It makes the blood to belong to the blood group. There are
aglutinogens on the erythrocyte membrane, which define the
blood group.
6 Participate in the maintenance of the metabolism of salts
and water. Erythrocytes can absorb the water on their
surface, increasing the osmotic pressure or ions, decreasing
the osmotic pressure.
7 Participate in homeostasis. Erythrocytes are the part of red
clot; they are a matrix for the formation of protrombinase.
Degraded erythrocytes co-operate in hypercoagulation and
formation of clot.

Common functional characteristics of erythrocytes

36
 I The amount of erythrocytes
There are 25•1012 erythrocytes in the blood of the human
organism. If we are to make the chain of all these erythrocytes,
then its length is going to be 200 000 km. This chain can
surround the Earth by equator 5 times.
The amount of erythrocytes in peripheral blood for male
is 4 - 5•1012/liter, for female is 3,5 - 4,5•1012/liter.
The decrease in amount of erythrocytes is erythropenia or
anemia. It can be absolute and relative.
Absolute erythropenia is the decrease in the total amount
of erythrocytes in the organism. Its reasons:
1) increase in the haemolysis of erythrocytes (due to exposure
to radiation, poisons, toxins, transfusion of the incompatible
blood etc.);
2) loss of blood;
3) decrease in speed or stopping of the erythropoiesis (because
of deficit of blood formation factors – iron, vitamins B 6, B12,
folic acid; because of erythropoietins deficiency when the
kidneys are pathologic; depression of blood formation
function of red bone marrow);
Relative erythropenia – decrease in erythrocytes amount
in blood volume unit when the blood is diluted. Its reasons:
 water retenssion in the in pathologies of the kidney;
 injecting the blood alternatives.
The increase of erythrocytes amount is erythrocytosis. It
can be absolute and relative.
Absolute erythrocytosis is the increase of the erythrocytes
amount in the organism. Its reason:
 increased of erythropoiesis because of the parcial
pressure in the air when in high altitude;
 because of great amount of erythropoietins during
hypoxia among the patients with chronic sickness of the
heart and lungs;

37
 because of leucocytosis.
Relative erythrocytosis is the increase in erythrocytes
amount in blood volume unit when the blood is concentrated.
Its reasons:
 much of sweating, nausea, diarrhea;
 scorches;
 shock;
 cholera, dysentery;
 hard muscle work (because of erythrocytes leaving the
spleen blood storage).

Methods of counting the amount of erythrocytes

1 Counting with the help of an autoanalyzer, based on
erythrocytes electroconduction.
2 Counting with celoscope, based on ability of erythrocytes to
absorb light.
3 Counting with Burker’s camera with Goryaev’s net.

II Shape of erythrocytes
Erythrocyte has a shape of biconcave disc, which when
cut transversely, looks like dumb-bells. This shape helps
erythrocytes to fulfill their main respiratory function.
This shape provides:
1) increase in the diffusion surface of erythrocyte.
With the help of this shape the internal surface of erythrocyte is
20% higher, than the one it can obtain when it is globe-shaped.
The total surface of all erythrocytes is 3800 m2; it is
1500 times larger, than the surface of human body.
2) shortening of diffusion distance. There is no
point inside the erythrocyte, which will be more far than 0.85
mkm from the surface. If the erythrocyte is round shaped, its
center would be in 2.5 mkm from the surface.
Variations in shape of red blood cells

38
 The following are the abnormal shape of red blood cells. Some of
these abnormal shapes of the red blood cells occur in different
types of anemia.
 Crenation: Shrinkage when in hypertonic solution.
 Spherocytosis: Globular form when in hypotonic solution.
 Elliptocytosis: Elliptical shape when in certain types of anemia.
 Sickle cell: Crescentic shape when in sickle cell anemia.
 Poikilocytosis: Unequal shapes due to deformed cell
membrane. The shape will be flask, hammer or any other
unusual shape.

III Diameter of erythrocytes

The average erythrocyte diameter is 7.5 mkm. The
allocation of erythrocytes in healthy human fits to normal
erythrocyte allocation or Praice-Jones curve (fig. 2.1).

number of
cells
120
Healthy
100

80

60 Perniciose anemia

40

20

1 3 5 7 9 11 13
diameter, mkm
Figure 2.1 - Curve of Praice-Jones.
In healthy human most of erythrocytes has diameter 7.5
mkm. There are also erythrocytes in blood of larger or smaller
diameter in blood, but they are not much. If there is

39
erythropoletic disorder then there are changes in Praice-Jones
curve. In macrocytosis, there is increase in the number of
erythrocytes, larger than 8 mkm (the diameter of some can
reach 12 mkm) – the curve moves to the right. In microcytosis,
there is the increase of the number of erythrocytes, smaller than
6 mkm (some can be even 2.2 mkm) – the curve moves to left.
In pernicios anemia there is poikilocytosis – the state when
erythrocytes of different shape are circulating in blood.
Variations in size of red blood cells
The size of the red blood cells alters in various conditions.
Microcytes are the red blood cells of small size and are present in
the following conditions:
 iron deficiency anemia;
 prolonged forced breathing and;
 increased osmotic pressure in blood.
Macrocytes are the red blood cells with larger size. The
macrocytes are present in the following conditions:
 megaloblastic anemia;
 muscular exercise and;
 decreased osmotic pressure in blood.

IV Erythrocyte is a non-nuclear cell

The loss of nucleus causes:
1) the increase of erythrocyte capacity (it is filled with
hemoglobin);
2) the decrease of oxygen usage. Erythrocytes is used an
amount of the oxygen, which is 200 times less than the cells
that form erythrocyte, that have nucleus. The erythrocyte,
supporting the whole body with oxygen, is using the less
part of it;
3) the absence of nucleus and the presence of elastic
membrane, allow erythrocyte to change its form easily to
pass through thin capillaries.

40
 V Plastic features of erythrocyte
It is the ability of the erythrocyte to change its shape.
Due to plastic features erythrocyte can pass through the
capillaries, which is twice thinner than erythrocyte itself. The
plastic features are provided by protein spectrin, which is
situated in the membrane and inside the erythrocyte itself.
Spectrin is 75% of all the proteins of erythrocyte. Its functions
are:
1) it forms the cytoskeleton and keeps the shape of
erythrocytes;
2) it gives membrane elastic features. Due to ability to contract
it helps erythrocytes to change their form.

VI Osmotic resistance of erythrocytes

It is the ability of erythrocyte membrane to resist osmotic
haemolysis.
The osmotic pressure inside the erythrocyte is a little
higher than osmotic pressure of blood plasma. That’s why
water is entering the erythrocyte, what provides normal turgor
of the cell. In hypotonic solution, water moves inside the
erythrocyte, resulting in swelling and rupture of the cell. It is
called osmotic haemolysis.
The measure of osmotic resistance is the concentration of
hypotonic solution, which causes haemolysis. In normal state
in human organism the destruction of the erythrocytes with the
smallest resistance begins in 0,54% solution of NaCl. This
value is called minimal osmotic resistance. When concentration
of NaCl is 0,42%, – 50% of erythrocytes are destructed, when
it is 0,34% – all erythrocytes are destructed. This value is
called maximal osmotic resistance (fig. 2.2).
During sickness (for example anemia) osmotic resistance
is decreased and haemolysis occurs in higher concentrations of
NaCl.

41
Figure 2.2 - Osmotic resistance of erythrocytes

VII Lifespan and fate of red blood cells

Average lifespan of red blood cell is about 120 days. The
senile red blood cells are destroyed in reticuloendothelial system.
When the cells become older (120 days), the cell membrane
becomes more and more fragile. The diameter of the capillaries is
less or equal to that of red blood cell. The younger red blood cells
can pass through the capillaries easily. However, because of the
fragile nature, the older cells are destroyed while trying to squeeze
through the capillaries. The destruction occurs mostly in the
capillaries of spleen because the splenic capillaries have a thin
lumen. So, the spleen is usually called ' graveyard of red blood
cells. The destroyed red blood cejls are"fragmented. From the
fragmented parts, the hemoglobin is released. The iron and globin
parts of the hemoglobin are separated with the production of
bilirubin. Iron combines with the protein-apoferritin to form
ferritin, which is stored in body. Globin also enters the protein
depot. The bilirubin is excreted by liver through bile. Daily 10%
red blood cells, which are senile, get destroyed in normal young
healthy adults. This causes release of about 0.6 g% of hemoglobin
into the plasma. From this 0.9 to 1.5 mg% bilirubin is formed.

42
 Determination of lifespan of red blood cell
The lifespan of the red blood cell is determined by
radioisotope method. The red blood cells are tagged with
radioactive substances like radioactive iron or radioactive
chromium. The life of red blood cell is determined by studying the
rate of loss of radioactive cells from circulation.

Erythrocyte sedimentation rate (ESR)

Erythrocytes can not sedimentate inside blood vessels.
Due to:
 constant blood movement;
 the charge of blood vessel wall and the charge of
erythrocyte is the same (negative) so cells repel from it.
If we put the blood inside the test-tube and add
anticoagulant than in few minutes we can observe the
sedimentation of erythrocytes, because the density of
erythrocytes (1,090 g/cm3) is higher, than the density of blood
plasma (1,025 – 1,034 g/cm3). The mechanism of the process is
as follows. At first erythrocytes form complexes with each
other (10-12 erythrocytes form “monetary column”). After
these complexes interact with plasma proteins, they become
heavier and they start to settle faster. Due to this process is not
equable in time (slow in the beginning, faster in the end) ESR
is determined for the fixed period of time, usually 1 hour.
In normal state ESR of men is 2-10 mm/hour, of women
2-15 mm/hour.

Factors, which affect ESR

The main mechanism of influence of all factors is the
changes in stickiness. There is inversed dependence between
stickiness and ESR (the higher stickness – the lower ESR, the
lower stickness – the higher ESR). That means that the factors,
which increase stickiness – they decrease ESR and otherwise.

43
The first group of factors – plasma factors:
1 The protein content of blood plasma
The influence of this factor is proved in the following
experiment. Erythrocytes of the patient with increased ESR are
put in blood plasma of the healthy man with the same blood
group. Erythrocytes of the patient sediments with normal
speed, otherwise erythrocytes of healthy man sediments in
patient’s blood plasma with higher speed.
Different proteins affect ESR in different ways. When
albumins concentration is increased, ESR decreases. When
concentration of high molecular proteins, globulins or
fibrinogen increases – ESR increases. Possibly, high molecular
proteins decrease electric charge on the erythrocytes
membrane, depress the electric repulsion of blood cells. Due to
this the aggregation properties of erythrocytes increase, ESR
increase. Globulins concentration increases in case of
inflammatory processes, infectious sicknesses and malignant
tumors. That is why these patients have increased level of ESR.
The amount of fibrinogen increases in 2 times in the
second half of pregnancy, that’s why before the delivery ESR
of the pregnant woman can reach 40 – 50 mm/hour.
2 Plasma volume
When increased plasma volume, hematocrit decreases,
blood stickiness decreases, and as a consequence ESR
increases.
The second group of factors – erythrocyte factors.
1 The amount of erythrocytes in blood volume
(hematocrit)
The higher amount of erythrocytes – the higher stickness
– the lower ESR.
The lower amount of erythrocytes – the lower stickness –
the higher ESR.
This is the reason of increase in ESR in anemic patients.
2 The ability of erythrocytes to aggregate

44
 The increase of erythrocyte ability to aggregate leads to
the decrease of stickiness, because the resistance of the
aggregates to friction is lower, than the resistance of separate
cells because of the decrease of correlation of the surface to the
volume. Aggregates sediments faster and ESR increases. The
increase of erythrocyte ability to aggregate is observed when
inflammatory processes and malignant tumors.
3 Erythrocytes shape
The change of the erythrocytes shape (for example when
sickle-cell anemia) or its modification (for example, when
pernicious anemia) can cause the oppression of the
erythrocytes ability to aggregate. It causes the increase of
stickiness and, as a consequence, the decrease of ESR.
Except these factors, there are some other ones, which
affect ESR. For example, steroid hormones (estrogen,
glucocorticoid hormones) and some medicine (salicylates)
increase ESR. Erythrocytes sedimentation rate increases when
the content of cholesterol in blood increases, during alkalosis,
and it decreases when content of bilious pigments and bilious
acids in blood increases and also during acidosis.

Functional properties of erythrocyte components

Components of erythrocyte are:
 membrane;
 fermentative systems;
 hemoglobin (Hb).
Erythrocyte membrane consists of bilipid layer, which
is bound with proteins. Membrane has plaques, which are filled
with glycoproteins and other proteins, carboxyl groups, which
form negative charge on the erythrocyte membrane, which is
called zeta-potential. The thickness of the membrane is 10
mkm. It is 1 million times more permeable for anions than for
cations. Anions of HCO3-, Cl-, and O2, CO2, H+ and OH- are
easily passing through it; Na+ and K+ are not passing.

45
 On the external side of the membrane there are sialic
acids and glycoproteins, which have antigenic properties and
define the blood group.
On the internal side of the membrane there are glycolytic
enzymes, Na-K-ATP-ase, glycoproteins, hemoglobin.
Fermentative systems of erythrocytes are represented
by:
 fermentative system of glycolysis;
 fermentative system of pentose cycle;
 glutationperoxydase fermentative system.
Metabolism of erythrocytes is different from other cells
metabolism.
At first, erythrocyte is using less than any other cell.
That’s why the amount of ATP formed is small. Mitochondria
is absent in erythrocyte and ATP is formed in glycolysis.
At second, metabolism is directed to maintain its ability
to bind oxygen, the recovery of iron ion in heme structure is
needed.
In the result of spontaneous oxidation of bivalent iron
Fe is transformed into trivalent iron Fe3+. And to bind the
2+

oxygen Fe3+ should be recovered to Fe2+ with the help of

NAD•H+.
Fermentative system of glycolysis supports erythrocyte
with:
 adenosintriphosphate (ATP);
 recovered NAD•H+, which is used for recovery of Fe3+
into Fe2+;
 2,3-biphosphoglycerate (2,3-BPG), is an important
intracellular regulator of hemoglobin functions.
2,3-BPG regulates the affinity of hemoglobin to the oxygen.
2,3-BPG connected to hemoglobin helps its deoxygenation.
Fermentative system of pentose cycle supports
erythrocyte with NADP•H+, which is the component of
antioxidative system and needed for the recovery of glutation.

46
 Glutationperoxidase system – is antioxidative system,
which protects a number of erythrocyte enzymes, which have
SH-group, from oxidation.
Hemoglobin (Hb) – is the main erythrocyte compound.
It makes 90% of the total solids of the cell. The fact that
hemoglobin is kept inside the cell is very important. If
hemoglobin was inside the blood plasma, it could cause a
number of disorders.
1 A large amount of free Hb does a toxic influence on
different tissues (neurons, kidneys).
2 In bloodstream Hb is turned to methemoglobin, but in the
erythrocyte there are fermentative systems, which predict
this to happen.
3 The amount of hemoglobin, needed for the transport of the
enough amount of oxygen will increase stickiness.
4 Hb will increase an oncotic pressure of plasma that will lead
to dehydration of tissues.
5 The part of Hb will be filtrated through the kidneys and it
will choke pores of kidney’s filter.

The structure of hemoglobin

Hemoglobin (Hb) – is a red pigment, chromoprotein,
which is situated in erythrocytes and transports oxygen.
There is 900 g of hemoglobin in human with body weight
70 kg. There are nearly 400 millions of hemoglobin molecules
in one erythrocyte. The molecular mass of hemoglobin is
64 450.
Hemoglobin has globular molecule, which is formed with
4 subunits. Each subunit contains heme. Heme – is Fe-
inclusive substance, the derivative of porphyrin. Heme
molecule consists of 4 pyrrol. The ion of Fe2+ is situated in the
center (fig. 2.3).

47
 Deoxygenated hemoglobin Oxygenated hemoglobin

Figure 2.3 – Types of hemoglobin

Heme is connected with a polypeptide. The complex of

polypeptides is called globin part of hemoglobin molecule
(globin). There are two pairs of polypeptide chains. Each chain
contains more than 140 amino acids. In dependence of number
and order of amino acids there are 4 types of chains: α, β, γ, δ
(α – 141, β – 146, γ – 146 amino acids) chains, each connected
to heme. Heme is disc shaped (fig.2.4).

Figure 2.4 – The schematic

image of hemoglobin A
There are

The main hemoglobin forms and compositions

Depending of protein chains there are following forms of
hemoglobin in normal state.
 Hb P (primitive) in embryo for the first 7-12 weeks
 Hb F (fetal) in fetus. Appears on ninth week. Consists of
2α- and 2γ- chains. Hb F can bind and transport oxygen

48
 easier (due to lesser similarity HbF to 2,3-BPG). That’s
why in the blood of fetus there is enough amount of
HbO2 formed, regardless lower tension of O2. Normally
after birth fetal hemoglobin is changed to adult
hemoglobin.
 HbA1 (adult). It contains 2α- and 2β- chains. HbA1 is
95% of all hemoglobin of adult.
 HbA2 – it contains 2α and 2δ chains. It is 5% of all
hemoglobin of adult.
In some inheritable diseases, there are defects of genes,
which encode α- or β- chains and the synthesis of Hb is
disturbed. These sicknesses are called thalassemias.
In α-thalassemia, the synthesis of α-chains is disturbed.
Erythrocytes are target-shaped, that’s why α-thalassemia is also
called target-shaped anemia. In β-thalassemias synthesis of β-
chains is disturbed (Kulee sickness).
Defects of the primary structure of hemoglobin also
belong to the pathological changes of hemoglobin. Mutative
genes, which produce abnormal hemoglobins, are widely
spread. There are a lot of forms of abnormal hemoglobins. For
example, if glutamate is changed to valine in β-chain,
pathological HbS is formed. In deoxygenated state its
dissolubility decreases 100 times, and it forms sediment. These
crystals deform erythrocyte. Erythrocyte gets sickle-shape,
hardly passes through small capillaries and phagocyted by
macrophages. It is called sickle-cell anemia.

Main physiological compositions of hemoglobin

1) HbO2 (oxygemoglobin) is the composition of Hb with
oxygen. It has red color, which define the red color of the
arterial blood.
Due to no oxidation occuring during interaction between
Hb and O2 and oxidation degree of iron does not change; the
reaction is called oxygenation (not oxidation).

49
 2) Hb (recovered Hb or deoxy Hb) – Hb, that releases O2. It
has cherry color, which defines the color of the venous blood.
Reaction of releasing the oxygen is called deoxygenation.
3) HbCO2 (carbhemoglobin) – the composition of Hb with
CO2.

Pathological composition of Hb:

1) HbCO (carboxyhemoglobin) – the composition of Hb with
CO.
Chemical relativity of Hb to CO is in 300 times higher
than O2. That’s why carbon monoxide displaces O2 from
hemoglobin, decreasing the ability of the blood to bind oxygen.
Even small number of CO leads to the significant increase in
formation of HbCO. When concentration of the CO in the air is
0,1% - 80% Hb binds not with O2, but with CO. When
concentration of CO in the air is 1%, in few seconds it will
cause death.
It is dangerous because HbCO is persistent and Hb
cannot transport oxygen anymore.
Low intoxication with CO is a reversible process and
after breathing fresh air, CO will gradually detach. Breathing
with clean oxygen has positive effect.
Normally HbCO is 1% of all the Hb. In smokers body it
is 3%, after heavy pull – 10%.
2) Met Hb (HbOH - methemoglobin) – hemoglobin, which
contains Fe3+ and has brown color. Oxidation of Fe2+ to Fe3+ in
hemoglobin occurs when interacting with strong oxidizers
(KMnO4, aniline), and also with medicine of oxidative
properties. Insignificant oxidation of hemoglobin to
methemoglobin also occurs in normal conditions. But with the
help of fermenting systems of erythrocyte (NADH-
methemoglobinreductase system) methemoglobin turns in to
hemoglobin. Inherited absence of this fermentation can cause
inherited methemoglobinemia.

50
 In pathological conditions, when methemoglobin is
formed, blood with high oxygen content circulates in the
organism, but it is not entering tissues.
The amount of hemoglobin in blood of healthy human is
140-160 g/L for men, 120-140 g/L for women, 200 g/L – for
newborns.

The definition of hemoglobin content:

1 Definition of the amount of bonded oxygen (1g of Hb can
bind 1,34 ml of O2).
2 Analysis of iron level in blood (iron content in hemoglobin
is 0,34%).
3 Tintometry (comparison of blood color with color of
standard solution) – Sali method.
4 Spectrophotometry.
Method 1 and 2 require sophisticated apparatus. Third –
is inaccurate. Fourth method is very popular nowadays. Blood
is mixed with the solution of potassium ferricyanide, potassium
cyanide, sodium bicarbonate. These substances will cause
destruction of erythrocytes; Hb turns to cyan methemoglobin
(HbCN). Unlike Hb, HbCN is stable and it can be stored for
few weeks. The solution is rayed with monochromatic light
with λ = 546nm, then extinction is defined. The content of Hb
is defined by special calibration scale.

T he follow
eryt forms of anemia:
1) Average hemoglobin content in one erythrocyte (AHC) –
characterizes the absolute number of Hb in the erythrocyte.

51
 AHC = Hb / E

Normally AHC = 26-36 picogram

When AHC is normal, than erythrocytes, they are called
normochromic.
When AHC is less than normal, erythrocytes are called
hypochromic.
When AHC is higher than normal, erythrocytes are called
hyperchromic.
2) Color index (CI) – the index which characterizes the
relative content of Hb in 1 erythrocyte.
CI = Hb / first three numbers in the amount of erythrocytes
Normally CI = 0,85 – 1,15
If CI is normal, erythrocytes (and anemia) are
normochromic. If CI is lower than normal – hypochromic. If CI
is higher than normal – hyperchromic.
3) Oxygen-carrying capacity of blood (OCC) – the amount
of oxygen, which is transported with 1 liter of blood
1 g of Hb can bind 1,34 ml of O2 - it is Hufner's number.

OCC = Hufner’s number • Hb (in g/l).

Regulation of erythrocyte content in peripheral blood

The erythrocyte content in peripheral blood of the adult is
3,5 – 5•1012/liter. The changes of this value (decrease or
increase) can lead to the dangerous changes in human
organism. The decrease in the erythrocyte amount leads to the
interruption of the oxygen transport in blood, which cause
ischemia of organs and tissues. The increase in the erythrocyte
amount is the reason of the increase in blood stickiness and the
increase of load on heart. When there is essential increase of
blood stickiness, the movement of the blood in vessels is
impossible.

52
Regulation of erythrocyte content is provided by regulation of
its formation (erythropoiesis) and destruction (haemolysis).

Variations in number of red blood cells

Physiological variations
I Increase in the red blood cell count is known as poly-
cythemia. If it occurs in physiological conditions, it is called
physiological polycythemia. It occurs in the following
conditions:
1) Age
At birth, the red blood cell count is 8 -10 millions/cu mm of
blood, The count decreases within 10 days after birth due to
destruction of cells causing physiological jaundice in some
infant. However, in infants and growing children, the cell
count is at a level higher than the value in adults.
2) Sex
Before puberty and after menopause in females the red blood
cell count is similar to that in males. During reproductive
period of females, the count is less than in males (4.5
millions/cu mm).
3) High Altitude
The inhabitants of mountains (above 10.000 feet from
mean sea level) have an increased red blood cell count of more
than 7 millions/cu mm. This is due to hypoxia in high altitude.
During hypoxia, the erythropoietin is released from the
kidneys. The erythropoietin in turn stimulates the bone marrow
to produce more red blood cells.
4) Muscular Exercise
There is a temporary increase in red blood cell count after
exercise. This is because of mild hypoxia and contraction of
spleen, which is the reservoir of blood.
5) Emotional Conditions
The red blood cell count is increased during the emotional
conditions like anxiety, because of sympathetic stimulation.

53
6) Increased Environmental Temperature
The increase in the atmospheric temperature increases red
blood cell count.
7) After Meals
There is a slight increase in the red blood cell count after taking
meals.
II Decrease in red blood cell count occurs in the follow-
ing physiological conditions:
1) High Barometric Pressures
At high barometric pressures as in deep sea, when the
oxygen tension of blood is higher, the red blood cell count
decreases.
2) After Sleep
The red blood cell count decreases slightly after sleep.
3) Pregnancy
In pregnancy, the red blood cell count decreases. This is
because of increase in extracellular fluid volume. Increase in
extracellular fluid volume, increases the plasma volume also resulting
in hemodilution. So, there is a relative reduction in the red blood cell
count.

Mechanisms of erythrocytes formation

Erythrocytes are formed in tissues of blood formation: in
the yolk sac of the embryo, in liver and spleen of the fetus and
in the red bone marrow of adult.
There are polypotent stem cells, which form all blood
cells.
There are 6 classes of red bone marrow cells:
1st class – stem cells (polypotent).
2nd class – half-stem cells (partially determined mediators of
hemopoesis).
3rd class – erythropoietin-sensitive cells (unipotent mediators of
hemopoesis). Cells of these classes are morphologically the
same.

54
4th class – blasts – erythroblast.
5th class – maturating cells (cells, which are differentiating) –
normoblast.
Two types of processes occur in maturating cells:
 Cells lose their organelles (nuclei, mitochondrias, and
endoplasmic reticulum)
 Synthesis and accumulation of hemoglobin occur.
Proerythroblast has large nucleus, it is characterized by
intensive proliferation (it divides every 8-12 hours).
Basophilic erythroblast – has smaller size, dyes with
basic stains. Hemoglobin appears in these cells.
Polychromatophilic erythroblast – has size, smaller than
basophilic, dyes with both basic and acidic stains.
Accumulation of hemoglobin occurs.
Oxyphilic erythroblast divides on the initial stage. Later
destruction of nuclei occurs, then nuclei disappear and cells
stop their division. They dye with acidic stains. The amount of
Hb increases.
6th class – mature cells (already differentiated) reticulocyte,
erythrocyte.
The amount of reticulocyte in the blood testifies about
the intensity of erythropoiesis. Normally its amount is 1% out
of all erythrocytes. The amount of reticulocytes increases when
activation of erythropoiesis. But in any case erythropoiesis can
be only 5-7 times more intensive comparably to the normal
level.
Due to absence of large erythrocytes depot in the
organism, liquidation of anemia after blood loss occurs with
the help of erythropoiesis. But the intensity of erythropoiesis in
red bone marrow starts after 3-5 days, and in peripheral blood
it is noticeable after 2-3 weeks.
Next factors of blood formation those are required for
erythropoiesis:

55
 Iron (for the synthesis of heme). The daily need of iron is
20-25 mg. 95% of this amount, organism gets from the
hemoglobin of destroyed erythrocytes and 5% (1mg) –
from food.
 Vitamin B12 – the external factor of blood formation.
Organism gets it from food, but it is absorbed only in
presence of internal factor of blood formation – Castle’s
factor, which is secreted by glands of the stomach.
 Folic acid. Gets into the organism from vegetative food.
Vitamin B12 and folic acid are required for the synthesis of
nucleic acids and globin.
 Vitamin C – participates in the metabolism of iron. Needed
in formation of heme, increases the activity of folic acid.
 Vitamin B6 – formation of heme.
 Vitamin B2 – formation of lipid part of the erythrocytes.
 Pantothenic acid – synthesis of phospholipids of the
erythrocytes membrane.

Mechanisms of the erythropoiesis regulation

1 Nervous:
 sympathetic nervous system stimulates erythropoiesis;
 parasympathetic – inhibits.
2 Humoral:
Somatotropic hormone, adrenocorticotropic hormone,
catecholamines, hormones of thyroid gland (thyroxin) and male
hormones stimulate erythropoiesis, female hormones – inhibit.
These nervous and humoral mechanisms are important
for blood formation. They work not directly but with the help
of specific mediators – “hormones of blood formation”.
Erythropoietin is a specific humoral stimulator of
erythropoiesis. 80% of erythropoietins are produced by
kidneys. Experimentally after nephrectomy the level of
erythropoietins will decrease. Synthesis of erythropoietins will
be inhibited in case of kidneys insufficiency. 20% of

56
erythropoietins are produced by macrophages. Hypoxia of
kidneys is a stimulator of erythropoietin production. There is a
protein, which can bind oxygen in the perivestibular cells of
kidneys. During sufficient oxygenation oxyform of
hemoprotein blocks gene, which is responsible for
erythropoietin synthesis and erythropoietin is not produced.
During hypoxia desoxyform (without oxygen) of hemoprotein
is produced, which cannot block this gene and synthesis of
erythropoietin occurs. Besides, when there is oxygen
insufficiency a number of enzymes that are sensitive to
hypoxia are activated in kidneys. Phospholipase A 2, which
supports the formation of prostaglandins, like prostaglandins E1
and E2, which activate adenylate cyclase and cause the increase
of cAMP concentration that increases synthesis and secretion
of erythropoietins.

Mechanisms of erythrocytes degradation

Mature erythrocytes circulate 100 - 120 days in the
bloodstream. After that they are engulfed by the cells of
reticuloendothelial system of the bone marrow, macrophages of
the liver and spleen. Small amount can go through haemolysis
in the bloodstream.
The main site of the erythrocyte degradation is the
spleen. Not only spleen, but any other tissue can degrade
erythrocytes, prove of this – is the fact that extravasation
gradually disappear in any part of the human body.
The reason of erythrocyte degradation (haemolysis) is
aging. Due to aging, the rate of metabolic processes decreases:
the activity of the enzymes of glycolysis and pentose phosphate
pathway decrease, as the consequence the number of ATP,
NAD•H and other important substances will decrease. The
consequences of decrease of the rate of metabolic processes
are:
1) elasticity loss: erythrocyte becomes unable to pass through
narrow site of the bloodstream and dwells there. One of
57
 these sites is spleen, where the distance between
trabeculations is less than 3 mkm. Erythrocytes dwell here,
the part of the cells and hemoglobin are engulfed by
macrophages;
2) the decrease in the ability to transform Fe3+ into Fe2+ (due to
insufficiency of NAD•H), which causes the disturbance of
gas transport function;
3) appearance of the sialic acids on the erythrocyte surface.
Macrophages have receptors to this groups that helps its co-
operation and degradation.
Engulfed erythrocytes go through haemolysis in the cells
of mononuclear phagocytic system (macrophages of spleen, red
bone marrow, Kupfer cells in liver). Hemoglobin is degraded
to heme and globin. Biliverdin is formed from heme. Biliverdin
forms bilirubin, which is called non-conjugated (it is insoluble
in water and reacts with diasoreagent only after treatment with
alcohol) and it is transported in the liver, where it interacts with
glucuronic acid. This bilirubin is called conjugated (it is
soluble in water and it reacts with diasoreagent). Conjugated
bilirubin and very small amount of non-conjugated is released
with bile into small intestine, where with the help of enzymes
of small intestine microflora, it turns into urobilinogen. There
are two possible ways of further transformation. The large part
of urobilinogen in the large intestine forms stercobilinogen,
which is excreted with faeces or with urine after absorption
into the bloodstream in the site of the superior and medium
hemorrhoid plexus of rectum. The lesser part of the
urobilinogen participates in hepato-intestinal circulation –
absorbed in the small intestine, gets to the liver, partially
oxidized and partially return to the intestine through bile ducts.

Forms of haemolysis

58
 The destruction of erythrocyte membrane accompanied
with the release of the hemoglobin into the blood plasma is
called haemolysis. Heamolyzed blood becomes transparent.
Depending on the reasons of degradation, there are other next
types of haemolysis:
1 Mechanical haemolysis. It is caused by the mechanical
degradation of the erythrocyte membrane. For example due to
ruin of the erythrocytes in the vessels of foot; or due to shaking
of glass with blood.
2 Osmotic haemolysis. It occurs when the osmotic
pressure inside the erythrocyte is higher than in blood plasma.
In this case, water due to laws of osmosis enters the
erythrocyte, its volume increases and the degradation of
membrane occurs. Reasons of osmotic haemolysis are:
 The decrease of the osmotic pressure of the medium,
where the erythrocyte is (hypotonic solution);
 The increase of the osmotic pressure in the erythrocyte
itself due to increase of the membrane permeability or
disturbance in work of Na-K pump.
3 Chemical haemolysis. It is haemolysis, which occur
under the influence of the substances that can degrade
erythrocyte membrane (ether, chloroform, alcohol, bilious
acids, saponine and others).
4 Thermal haemolysis. It is haemolysis, which is caused
by the influence of high or low temperatures. For example
during deep-freeze of the blood.
5 Biological haemolysis. It is haemolysis, which develops
after transfusion of incompatible blood and after sting of some
snakes.

Questions for self-control

1 The definition of erythron.
2 Functions of erythrocytes.

59
3 The amount of erythrocytes. The definition of erythrocytosis
and erythropenia.
4 Methods of calculating erythrocytes.
5 The shape of erythrocytes.
6 Diameter of erythrocytes. Praice-Jones curve.
7 Plasticity of erythrocytes.
8 Osmotic resistance of erythrocytes.
9 Erythrocyte sedimentation rate (ESR). Factors, which affect
ESR.
10 Functional properties of erythrocyte elements.
11 Forms and compositions of hemoglobin.
12 Methods of determination of hemoglobin content in
peripheral blood.
13 Values, which are used for erythropoiesis diagnostics.
14 The formation of erythrocytes in the organism.
15 Mechanisms of erythropoiesis regulation.
16 Reasons and mechanisms of erythrocytes degradation.
17 Forms of haemolysis.

Tests for self-control

1 Which value shows relative content of hemoglobin in every
single erythrocyte:
a) erythrocyte sedimentation rate (ESR) ;
b) hematocrit;
c) diameter of erythrocytes;
d) Praice-Jones curve;
e) color index;
f) no correct answer?

2 Reticulocytes are immature forms of:

a) erythrocytes;
b) lymphocytes;
c) neutrophils;
d) monocytes;

60
 e) thrombocytes;
f) basophils;
g) eosinophils;
h) no correct answer.
3 Name one organ where erythrocytes go through
physiological degradation:
a) red bone marrow;
b) lymphatic nodules;
c) liver;
d) spleen;
e) lungs;
f) kidneys;
g) thymus;
h) no correct answer.

4 When does hematocrit decrease:

a) increase of erythrocyte content in blood;
b) decrease of erythrocyte content in blood;
c) decrease of plasma volume;
d) increase of plasma volume;
e) no correct answer?

5 What type of haemolysis will cause hypotonic solutions:

a) chemical;
b) biological;
c) osmotic;
d) thermal;
e) no correct answer?

6 What is the normal value for erythrocyte sedimentation rate

in male:
a) 0 – 1%;
b) 1 – 10%;
c) 2 – 10 mm/hour;

61
 d) 2 – 15 mm/hour;
e) 7 – 15 mm/hour;
f) no correct answer?

7 What cause the increase in ESR:

a) increase in the number of erythrocytes;
b) decrease in the number of erythrocytes;
c) increase in the number of globulins;
d) decrease in the number of globulins;
e) increase in the number of albumins;
f) decrease in the number of albumins?

8 How long do erythrocytes circulate in the bloodstream:

a) 2 – 3 months;
b) 5 – 6 months;
c) 100 – 120 days;
d) 40 – 50 days;
e) 1 year;
f) no correct answer?

9 What are the factors, which increase erythropoiesis:

a) androgens;
b) estrogens;
c) somatotropic hormone;
d) adrenalin;
e) thyroxin;
f) no correct answer?

10 What is the main function of erythrocytes:

a) respiratory;
b) protection;
c) immune;
d) trophic;
e) energetic;

62
 f) no correct answer?

11 What is oxygen-carrying capacity of the patient, if his

hemoglobin level is 100 g/l:
a) 100 ml;
b) 125 ml;
c) 134 ml;
d) 146 ml;
e) no correct answer?

12 What are non-nuclear cells of the blood:

a) lymphocytes;
b) monocytes;
c) basophiles;
d) neutrophils;
e) erythrocytes;
f) no correct answer?

13 If concentration of 2,3-DPG will increase, what will happen

to the amount of the oxygen, transported with hemoglobin:
a) decrease;
b) increase;
c) will not change?

14 What is the composition of hemoglobin with carbon

dioxide:
a) oxyhemoglobin;
b) deoxyhemoglobin;
c) carboxyhemoglobin;
d) carbhemoglobin;
e) methemoglobin;
f) no correct answer?

15 How many hemes are in one molecule of hemoglobin:

63
a) 1;
b) 2;
c) 3;
d) 4;
e) 8?

64
CHAPTER 3 Blood groups

The definition of agglutinogens,

agglutinins, agglutination

Agglutinogens (hemagglutinogens) – specific antigens,

which are situated on the erythrocyte membrane. By chemical
nature they are glycoproteins or glycolipids. There is an
individual set of specific erythrocyte agglutinogens.
Approximately 400 are discovered now. Thirty are mostly
common and they can be a reason of reactions after blood
transfusion.
Agglutinins (isohemagglutinins) – specific antibodies,
which are dissolved in blood plasma, they are related to γ-
globulins fraction.
Agglutination – is agglutination of erythrocytes, which
occur in the result of reaction of antigen-antibody. As a rule,
agglutination is accompanied by erythrocyte haemolysis.
Agglutination occurs after similar agglutinogens meet
agglutinins. Agglutinin has 2 active centers, which are why it
can bind 2 erythrocytes by forming a “bridge” between them.
In 1901 austrian K. Landsteiner and in 1903 czech Y.
Yanskiy found agglutinogens A and B on the surface of the
erythrocytes and explained the agglutination.
Definition of the blood group by ABO system is based on
the presence of aggluttinogens A and B on the surface of the
erythrocytes. Agglutinins α and β are formed during the first
year of life. Agglutinins are formed to agglutinogens which are
absent on the erythrocyte surface (if erythrocytes have
agglutinogen A, agglutinin β is formed, if B – α). Agglutinins
relate mostly to immunoglobulins M (IgM). They are high-
molecular immunoglobulins. IgM are typical hemolysins
(when they interact with relative antigens on the erythrocyte
membrane, they form substances, which destroy erythrocytes).

65
 If similar agglutinogens and agglutinins meet: A with α,
B with β – agglutination occurs, which ends with haemolysis
of erythrocytes. Lysis of erythrocytes performs with the help of
complement system and proteolytic enzymes. Accumulation of
destroyed erythrocytes leads to obstruction of capillaries and
other complications, which can cause death. That’s why in
natural conditions human organism cannot have antigens and
antibodies, which are relatied to each other, because it could
lead to agglutination of own erythrocytes.

There are 4 blood groups in ABO system:

Blood Agglutinogens Agglutinins

group (erythrocytes) (plasma)
І (О) – α, β
ІІ (А) А β
ІІІ (В) В α
ІV (АВ) АВ –

Current ideas about ABO blood groups

Two particular types of antigens are much more likely
than the others to cause blood transfusion reactions. They are
the O-A-B system of antigens and the Rh system.
Four major O-A-B blood types, as shown in table above,
depending on the presence or absence of the two
agglutinogens, the A and B agglutinogens.When neither A nor
B agglutinogen is present, the blood is type O. When only type
A agglutinogen is present, the blood is type A.When only type
B agglutinogen is present, the blood is type B.When both A and
B agglutinogens are present, the blood is type AB. These
combinations of genes are known as the genotypes, and each
person is one of the six genotypes. When type A agglutinogen
is not present in a person’s red blood cells, antibodies known
as anti-A agglutinins develop in the plasma. Also, when type B

66
agglutinogen is not present in the red blood cells, antibodies
known as anti-B agglutinins develop in the plasma. Thus, note
that type O blood, lthough containing no agglutinogens, does
contain both anti-A and anti-B agglutinins; type A blood
contains type A agglutinogens and anti-B agglutinins; type B
blood contains type B agglutinogens and anti-A agglutinins.
Finally, type AB blood contains both A and B agglutinogens
but no agglutinins.

Titre of the Agglutinins at Different Ages

Immediately after birth, the quantity of agglutinins in the

plasma is almost zero. Two to 8 months after birth, an infant
begins to produce agglutinins—anti-A agglutinins when type A
agglutinogens are not present in the cells, and anti-B
agglutinins when type B agglutinogens are not in the cells.
Figure 3.1 shows the changing titres of the anti-A and anti-B
agglutinins at different ages. A maximum titre is usually
reached at 8 to 10 years of age, and this gradually declines
throughout the remaining years of life.

67
Figure 3.1 – Average titers of anti-A and anti-Baglutinins in the plasmas of
piples with different blood types.

Relative rates of red blood cell production in the bone

marrow of different bones at different ages.

Origin of Agglutinins in the Plasma

The agglutinins are gamma globulins, as are almost all

antibodies, and they are produced by the same bone marrow
and lymph gland cells that produce antibodies to any other
antigens. Most of them are IgM and IgG immunoglobulin
molecules.
But why are these agglutinins produced in people who do
not have the respective agglutinogens in their red blood cells?
The answer to this is that small amounts of type A and B
antigens enter the body through food, through bacteria, and
other ways, and these substances initiate the development of
the anti-A and anti-B agglutinins. For instance, infusion of
group A antigen into a recipient having a non-A blood type
causes a typical immune response with formation of greater
quantities of anti-A agglutinins than ever.Also, the neonate has
few, if any, agglutinins, showing that agglutinin formation
occurs almost entirely after birth.

Agglutination Process In Transfusion Reactions

When blood is mismatched so that anti-A or anti-B

plasma agglutinins are mixed with red blood cells that contain
A or B agglutinogens, respectively, the red cells agglutinate as
a result of the agglutinins’ attaching themselves to the red
blood cells (Fig. 3.2). Because the agglutinins have two binding
sites (IgG type) or 10 binding sites (IgM type), a single
agglutinin can attach to two or more red blood cells at the same
time, thereby causing the cells to be bound together by the

68
agglutinin. This causes the cells to clump, which is the process
of “agglutination.” Then these clumps plug small blood vessels
throughout the circulatory system. During ensuing hours to
days, either physical distortion of the cells or attack by
phagocytic white blood cells destroys the membranes of the
agglutinated cells, releasing hemoglobin into the plasma, which
is called “hemolysis” of the red blood cells.

Type Type

Antigens on
red blood
cells

Antibodies
in plasma

Agglutinatio
n reaction

Figure 3.2 Agglutination reaction. People with type A blood have

type A antigens on their red blood cells and antibodies in their plasma
against the type B antigen. People with type B blood have type B antigens
on their red blood cells and antibodies in their plasma against the type A
antigen. Therefore, if red blood cells from one blood type are mixed with
antibodies from the plasma of the other blood type, an agglutination
reaction occurs. In this reaction, red blood cells stick together because of

69
antigen-antibody binding. Acute hemolysis occurs in some transfusion
reactions.

Sometimes, when recipient and donor is blood is

mismatched, immediate hemolysis of red cells occurs in the
circulating blood. In this case, the antibodies cause lysis of the
red blood cells by activating the complement system, which
releases proteolytic enzymes (the lytic complex) that rupture
the cell membranes. Immediate intravascular hemolysis is far
less common than agglutination followed by delayed
hemolysis, because not only does there have to be a high titre
of antibodies for lysis to occur, but also a different type of
antibody seems to be required, mainly the IgM antibodies;
these antibodies are called hemolysins.
Human’s blood group is defined by the antigen properties
of erythrocytes. Antigens A and B are glycoproteins, which
consist 75% of carbohydrates, 15% of amino acids and 10% of
phospholipids. Antigen properties depend on the nature of the
sugar in glycoproteins, in other words peculiarity of antigen is
defined by its carbohydrate part.
Patients with O-group (I) have antigen H, which has
three carbohydrate tails (N-acetylgalactosamine, galactose,
fucose).
Erythrocytes of II group have fourth tail, connected to
previous - N-acetylgalactosamin.
Erythrocytes of III group have fourth tail, connected to
previous - galactose.
In erythrocytes of IV group part of glycoproteins ends
with galactose and part ends with N-acetylgalactosamin (fig.
3.3).
There are two sites in genome of the cell, which is
responsible for blood formation – H and ABO, which are
responsible for the synthesis of gene.

70
Figure 3.3 - Structure of glycoprotein of the erythrocyte membrane

In H-site one antigen is formed:

 H-gene, which encodes enzyme fucosyl-transferase that
transports fucose to galactose and controls synthesis of antigen
H.
In ABO-site three antigens are formed:
 Gene-O, which encodes protein that does not have
fermentation activity.
 A-gene, which encodes enzyme A specific transferase that
transports N-acetylgalactosamine to fucose, subsequently
antigen A is formed on the erythrocyte membrane.

71
 B-gene, which encodes enzyme B specific transferase that
transports galactose to fucose, subsequently antigen B is
formed on the erythrocyte membrane.

Definition of ABO blood group system

The same principle is used to define the blood group in

every system: to provide conditions for the agglutination of
erythrocytes in the medium of standard isohemagglutinating
serums or coliclones, which have high titre of antibodies for
the examined erythrocytes antigens.
Standard serum is a prepared blood plasma of donors
with different blood groups, without fibrinogen and has high
concentration of antibodies for one or several antigens of one
blood group.
There are 4 groups of serums in ABO system. The serum
of group I has agglutinins α and β (colorless); group II –
agglutinins β (light blue color); group III – agglutinins α (pink
color); IV group – does not have agglutinins (yellow color).
Coliclones is a powder, which includes specific
immunoglobulins (antibodies) that work against group
antigens. These antibodies are formed by monoclonal B-
lymphocytes in mouse after injecting in its organism antigens
in the form of malignant specific cells.
Coliclones have antibodies with only one peculiarity.
That means, they react with only one antigen i. e. they do not
lead to non-specific polyagglutination of erythrocytes. This
property makes them more preferable than standard serums. It
is hard to clear standard serums from other antibodies and that
is why non-specific reactions with antigen of the examined
blood are possible.
There are 2 coliclones in ABO blood system: anti-A and
anti-B.
Serums (or coliclones) are mixed on the board with blood

72
in correlation 10:1. Observer is looking after reaction for 2,5
minutes. Drops of serum with agglutination will become
transparent and erythrocytes gather in masses.

According to agglutination test the blood group is defined

serum І ІІ ІІІ ІV
blood (αβ) (β) (α) (-)

І (0) – – – –
ІІ ( А) + – + –
ІІІ ( В) + + – –
ІV( АВ) + + + –

Rhesus system

In 1940 K. Landsteiner and I. Winner found one more

antigen on the erythrocytes of macaque rhesus and they named
it rhesus-factor. Later it was discovered that 85% of all whites
has it, and their blood is called rhesus-positive. 15% of humans
do not have this gene and their blood is rhesus-negative. In
American blacks, the percentage of Rh positives is about 95,
whereas in African blacks, it is virtually 100 per cent.
There are six common types of Rh antigens, each of
which is called an Rh factor. These types are designated C, D,
E, c, d, and e.A person who has a C antigen does not have the c
antigen, but the person missing the C antigen always has the c
antigen. The same is true for the D-d and E-e antigens. Also,
because of the manner of inheritance of these factors, each
person has one of each of the three pairs of antigens. The type
D antigen is widely prevalent in the population and

73
considerably more antigenic than the other Rh antigens.
Anyone who has this type of antigen is said to be Rh positive,
whereas a person who does not have type D antigen is said to
be Rh negative. However, it must be noted that even in Rh-
negative people, some of the other Rh antigens can still cause
transfusion reactions, although the reactions are usually much
mild.

There are 2 blood groups in rhesus system:

+
Rh - blood has antigen D;
Rh- - blood does not have antigen D.

The difference between rhesus and ABO system

1 Agglutinins of ABO system appear on the first months of

life and stay in blood for the entire life. Antirhesus-antibodies
appear only after sensitization (contact of Rh - patient with Rh+-
antigens). This can occur after blood transfusion and during
pregnancy.
2 Rh-agglutinins – are insufficient antibodies of IgG class,
they have small sizes and they can pass through the placental
barrier. Agglutinins α and β are sufficient antibodies of IgM
class; they have big sizes and cannot pass through placenta.

Rhesus conflict

Rhesus conflict can occur in 2 cases.

1 After blood transfusion (after transfusion of Rh+ blood
to Rh- recipient). The first blood transfusion is not dangerous.
The maximal titre of antibodies is after 2-4 months. After this
period, transfused erythrocytes are derived out of bloodstream.
But antirhesus-antibodies are already in the blood of the
patient. And after the second transfusion of Rh+ blood
agglutination occurs, haemolysis of erythrocytes, which can

74
lead to hemolytic shock and death.
Formation of Anti-Rh Agglutinins
When red blood cells containing Rh factor are injected
into a person whose blood does not contain the Rh factor—that
is, into an Rh-negative person—anti-Rh agglutinins develop
slowly, reaching maximum concentration of agglutinins about
2 to 4 months later.This immune response occurs to a much
greater extent in some people than in others. With multiple
exposures to the Rh factor, an Rh-negative person eventually
becomes strongly “sensitized” to Rh factor.
Characteristics of Rh Transfusion Reactions
If an Rh-negative person has never been exposed to Rh-
positive blood, transfusion of Rh-positive blood into that
person will likely cause no immediate reaction (fig 3.4).

First transfusion Second transfusion

Transfusion of Formation of Agglutination of

Rh+ erythrocytes antirhesus- erythrocytes after the
to Rh- recipient antibodies in second transfusion
recipient’s blood
Figure 3.4 - Rhesus conflict of Rh Transfusion Reactions

However, anti-Rh antibodies can develop in sufficient

quantities during the next 2 to 4 weeks to cause agglutination
of those transfused cells that are still circulating in the
blood.These cells are then hemolyzed by the tissue macrophage

75
system. Thus, a delayed transfusion reaction occurs, although it
is usually mild. On subsequent transfusion of Rh-positive
blood into the same person, who is now already immunized
against the Rh factor, the transfusion reaction is greatly
enhanced and can be immediate and as severe as a transfusion
reaction caused by mismatched type A or B blood.

2 During pregnancy (if Rh- woman is pregnant with

+
Rh fetus). As usual there are no complications during first
pregnancy. During delivery placental barrier is disturbed and
erythrocytes of fetus get into the blood of woman. The
formation of antirhesus-antibodies (IgG) starts.

First pregnancy Formation of

Rh+ erythrocytes Second pregnancy
Antirhesus-antibodies
enters woman’s antirhesus- enters fetus’s organism,
organism during antibodies in haemolysis of Rh+
delivery woman’s blood erythrocytes of fetus

Figure 3.5 – Resus conflict by pregnancy

During second Rh-conflict pregnancy, antirhesus-

antibodies are passing through placenta in the organism of etus

76
and it will cause the destruction of erythrocytes, which will
cause the death of the fetus and missbirth. If during first
pregnancy there is fetoplacental insufficiency, small amount of
erythrocytes can get into the woman’s organism and cause the
production of immunoglobulins. As usual, titre of antibodies
increases slowly during several months that are why no serious
complications occur. In this case hemolytic anemia of newborn
can take place (fig. 3.5).
Erythroblastosis Fetalis “Hemolytic Disease of the
Newborn”
Erythroblastosis fetalis is a disease of the fetus and
newborn child characterized by agglutination and phagocytosis
of the fetus’s red blood cells. In most instances of
erythroblastosis fetalis, the mother is Rh negative and the
father Rh positive. The baby has inherited the Rh-positive
antigen from the father, and the mother develops anti-Rh
agglutinins from exposure to the fetus’s Rh antigen. In turn, the
mother’s agglutinins diffuse through the placenta into the fetus
and cause red blood cell agglutination.
Incidence of the Disease
An Rh-negative mother having her first Rh-positive child
usually does not develop sufficient anti-Rh agglutinins to cause
any harm. However, about 3 per cent of second Rh-positive
babies exhibit some signs of erythroblastosis fetalis; about 10
per cent of third babies exhibit the disease; and the incidence
rises progressively with subsequent pregnancies.
Effect of the Mother’s Antibodies on the Fetus
After anti- Rh antibodies have formed in the mother, they
diffuse slowly through the placental membrane into the fetus’s
blood. There they cause agglutination of the fetus’s blood. The
agglutinated red blood cells subsequently hemolyze, releasing
hemoglobin into the blood. The fetus’s macrophages then
convert the hemoglobin into bilirubin, which causes the baby’s

77
skin to become yellow (jaundiced).The antibodies can also
attack and damage other cells of the body.
Clinical Picture of Erythroblastosis
The jaundiced, erythroblastotic newborn baby is usually
anemic at birth, and the anti-Rh agglutinins from the mother
usually circulate in the infant’s blood for another 1 to 2 months
after birth, destroying more and more red blood cells. The
hematopoietic tissues of the infant attempt to replace the
hemolyzed red blood cells. The liver and spleen become
greatly enlarged and produce red blood cells in the same
manner that they normally do during the middle of gestation.
Because of the rapid production of red cells, many early forms
of red blood cells, including many nucleated blastic forms, are
passed from the baby’s bone marrow into the circulatory
system, and it is because of the presence of these nucleated
blastic red blood cells that the disease is called rythroblastosis
fetalis. Although the severe anemia of erythroblastosis fetalis is
usually the cause of death, many children who barely survive
the anemia exhibit permanent mental impairment or damage to
motor areas of the brain because of precipitation of bilirubin in
the neuronal cells, causing destruction of many, a condition
called kernicterus.
Treatment of the Erythroblastotic Neonate
One treatment for erythroblastosis fetalis is to replace the
neonate’s blood with Rh-negative blood. About 400 milliliters
of Rh-negative blood is infused over a period of 1.5 or more
hours while the neonate’s own Rh-positive blood is being
removed. This procedure may be repeated several times during
the first few weeks of life, mainly to keep the bilirubin level
low and thereby prevent kernicterus. By the time these
transfused Rh-negative cells are replaced with the infant’s own
Rh-positive cells, a process that requires 6 or more weeks, the
anti- Rh agglutinins that had come from the mother will have
been destroyed.

78
 The formation of antibodies in the organism of Rh- -
woman can be depressed or totally inhibited with the help of
anti D-prophylaxis. Immediately after delivery anti D-globulin
is injected into woman’s organism. Rh+ erythrocytes, which got
into her blood, will be destroyed. In this way the factor, which
should cause synthesis of antibodies will be terminated.
By the way, reaction antigen-antibody can occur when there is
incompatibility of ABO-blood system. But these reactions have
low degree of expression. Incompatibility of blood group by
ABO system of mother and fetus can predict sensitization,
which occur when there is incompatibility of Rh system.
Erythrocytes of fetus are removed by α and β – agglutinins and
Rh-factor is no longer able to activate immune system of
mother. That is why in cases, when Rh - women can normally
give birth to more than one child, are often.

Other systems of blood

In 1930 for the discovery of blood groups K. Landsteiner

was granted with the Nobel Prize. During ceremony he said
that new agglutinogens will be discovered in future and the
quantity of blood groups will increase until it reaches the
number of Earth’s population. And he was right. Even in ABO
blood system several variants of every agglutinogen was
found. There are 10 variants of agglutinogen A. The difference
between them is that A1 is the most active, and others have less
antigenic properties. The blood group of these patients can be
mistakenly defined as 1 blood group that can cause
complications during transfusion.
Except ABO blood system, the most important are Rh, MNS,
P, Luteran, Kell-Chelano, Daffi, Diego, and Kid. Antigens of
these blood systems are situated on erythrocytes, independently
on ABO system and independently on each other. These
systems are important only when there are often blood

79
transfusions and during pregnancy, which is incompatible by
any of these antigens.

Blood transfusion

It should be mentioned that blood transfusion is the

transplantation of non-self tissue. And the first of
complications is the immune conflict. The doctor, who made
this operation, has the whole responsibility for the
consequences, so he should be carefully watching after every
stages of it.
The stages of blood transfusion:
First stage: The definition of donor’s and recipient’s
blood group by ABO and Rh systems.
Second stage: Individual compatibility test. Its objective
is to check compatibility for other antigens and antibodies.
Straight test – donor’s erythrocytes + recipient’s blood
plasma. This test allows finding antibodies in recipient’s serum
for donor’s erythrocytes.
Reverse test – donor’s blood plasma + recipient’s
erythrocytes. This test allows finding antibodies in donor’s
blood for recipient’s erythrocytes.
Absence of agglutinations in both tests confirms
compatibility.
Third stage: Biological test.
Its objective: proteins compatibility test. 25 ml of donor’s
blood is injected intravenously into the recipient and reaction
of the patient is observed.
Complications for the blood transfusion are pains in the
lumbal region, darkening of the eyes, increase in body
temperature.
If blood is transfused into the patient without conscience,
then blood pressure, pulse and pupils are observed. Blood
pressure should not change; pupils should be in normal state.

80
The increase in blood pressure for 20 mmHg is the signal for
the transfusion stoppage.

Rules of blood transfusion

1) The blood should have the same group and same rhesus.
Before, people with I(O) blood group are thought to be
universal donors. Their blood was used to transfuse to the
recipients of other groups. Now these blood transfusions are
not allowed. Why? Erythrocytes of I group do not have
antigens A and B and in fact packed red cells of I group can
be transfused to the recipients of other group. But plasma of
I blood group includes agglutinins α and β, that’s why it can
cause agglutination in blood, which includes agglutinogens
A and B (blood with II, III and IV group). This plasma can
be injected to the recipients in limited quantities, so
transfused agglutinins will be dissolved and agglutination
will not occur.
2) Blood should not be injected more than 500 ml of blood at
once.
3) Blood should not be transfused from the same donor more
than once.

Indication of Blood Transfusion:

 Decreased blood volume (20% is lost):e.g. in
haemorrhage.
 Decreased R. B. Cs.in anemia when Hb % is below 40%.
 Decreased W. B. Cs. (leucopenia).
 Decreased blood platelets (thrombocytopenic purpura).
 Decreased coagulation factors VIII, IX, and XI in
haemophilia.
 In erythroblastosis fetalis: exchange transfusion to replace
the infant’s blood with Rh-ve group O blood.

Dangers of blood transfusion:

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 I Dangers of Incompatibility:
1) agglutination (clamping) of the donor”s R.B.Cs. This:

a) blocks the capillaries and causes severe pain;

b) Blocks the blood vessels, e.g. of the heart causing
myocardial infarction or thr brain causing paralysis.
2) hemolysis of the donor’s R.B.Cs. leads to jaundice due to
excess formation of bilirubin;
3) hypotension: fall of arterial blood pressure due to V.D.
caused by histamine released from hemolysed R.B.Cs;
4) decreased urine volume (oliguria) or even anuria and death
may occur from renal failure, caused by:
a) hypotension;
b) precipitation of Hb (acid hematin) and blocking of
renal tubules causing anuria;
c) fatal hyperkalaemia may result from hemolyssis of
R.B.Cs. and failure of K + excretion due to renal failure;
d) fatal hypokalaemia, may occur during recovery, since
K filtered from the glomeruli is not reabsorbed by
damaged renal tubules.
II Transmsssion of Diseases:
As syplilis, malaria, viral hepatitis or AIDS.
III Allergic reactions:
Rigors and fever may occur due to presence of pyrogens.
IV Transmsssion of excessive amount of blood:
This may cause an overload and leads to heart failure.
V Tetany:
Increased neuromuscular excitability due to decreased calcium
level in plasma. It may occur due to excessive amount of
citrate in the transfused blood.

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 Questions for self-control
1 Definition of agglutinogens, agglutinins, agglutination.
2 The characteristics of blood groups by ABO system.
3 Modern ideas about blood groups of ABO system.
4 The definition of blood groups by ABO system.
5 The characteristics of blood groups by rhesus system.
6 The definition of rhesus-conflict.
7 Other blood systems.
8 Stages of blood transfusion.
9 Rules of blood transfusion.

Tests for self-control

1 Erythrocytes of which blood group don’t have agglutinogens
A on their surface:
a) group I;
b) group II;
c) group III;
d) group IV;
e) there is no such a blood group.

2 Erythrocytes of which blood group have agglutinogens B on

their surface:
a) group I;
b) group ;
c) group III;
d) group IV;
e) there is no such a blood group.

3 Blood plasma of which group has agglutinins anti-A:

a) group I;
b) group II;
c) group III;
d) group IV;
e) there is no such a blood group.

83
4 Blood plasma of which group has agglutinins anti-A and
agglutinins anti-B:
a) group I;
b) group II;
c) group III;
d) group IV;
e) there is no such a blood group.

5 Blood plasma of which group doesn’t have agglutinins anti-

B:
a) group I;
b) group II;
c) group III;
d) group IV;
e) there is no such a blood group.

6 During the blood test of patient’s blood by ABO system

agglutination occurred in standard serums of I and III blood
group. Name the patient’s blood group:
a) group I;
b) group II;
c) group III;
d) group IV;
e) inaccuracy occurred and test should be repeated.

7 During the blood test of patient’s blood by ABO system

agglutination of erythrocytes did not occur in any of three
standard serums (group I, II, III). Name the patient’s blood
group:
a) group I;
b) group II;
c) group III;
d) group IV;
e) inaccuracy occurred and test should be repeated.

84
8 Blood group I in ABO system is characterized by presence
of:
a) agglutinogens A;
b) agglutinogens B;
c) agglutinins anti-A;
d) agglutinins anti-B;
e) no correct answer.

9 Blood group II in ABO system is characterized by the

absence of:
a) agglutinogens A;
b) agglutinogens B;
c) agglutinins anti-A;
d) agglutinins anti-B;
e) no correct answer.

10 In what cases is blood transfusion not allowed:

a) donor’s blood group is O(I), recipient’s blood group is
A(II);
b) donor’s blood group is A(II), recipient’s blood group is
B(III);
c) donor’s blood group is B(III), recipient’s blood group is
AB(IV);
d) donor’s blood group is AB(IV), recipient’s blood group
is O(I);
e) donor’s blood group is O(I), recipient’s blood group is
AB(IV);
f) is allowed in all cases?

11 In what cases is blood transfusion allowed:

a) donor’s blood group is B(III), recipient’s blood group is
B(III);
b) donor’s blood group is A(II), recipient’s blood group is
A(II);

85
 c) donor’s blood group is B(III), recipient’s blood group is
AB(IV);
d) donor’s blood group is AB(IV), recipient’s blood group
is O(I);
e) donor’s blood group is O(I), recipient’s blood group is
AB(IV);
f) no correct answer?

12 Where are agglutinogens A located:

a) in cytoplasm of erythrocytes;
b) on the surface of the erythrocytes membrane;
c) in blood plasma;
d) in cytoplasm of leucocytes;
e) in the nucleus of leucocyte;
f) inside the thrombocytes;
g) no correct answer?

13 Where are agglutinins anti-A located:

a) in cytoplasm of erythrocytes;
b) on the surface of the erythrocytes membrane;
c) in blood plasma;
d) in cytoplasm of leucocytes;
e) in the nucleus of leucocytes;
f) inside the thrombocytes;
g) no correct answer?

14 What do erythrocytes of Rh+ (rhesus positive) blood have on

their surface:
a) A-antigen;
b) B-antigen;
c) C-antigen;
d) D-antigen;
e) E-antigen;
f) G-antigen;

86
 g) K-antigen;
h) no correct answer?

15 In which of the following cases of blood transfusion there is

danger in the life of the patient:
a) if Rh+ blood is transfused to the Rh+ patient;
b) if Rh- blood is transfused to the Rh+ patient;
c) if Rh+ blood is transfused to the Rh- patient;
d) if Rh- blood is transfused to the Rh- patient;
e) there is no danger in these cases?

87
CHAPTER 4 Protective functions of the blood. Leucocytes

Our bodies are exposed continually to bacteria, viruses,

fungi, and parasites, all of which occur normally and to varying
degrees in the skin, the mouth, the respiratory passageways, the
intestinal tract, the lining membranes of the eyes, and even the
urinary tract. Many of these infectious agents are capable of
causing serious abnormal physiological function or even death
if they invade the deeper tissues. In addition, we are exposed
intermittently to other highly infectious bacteria and viruses
besides those that are normally present, and these can cause;
acute lethal diseases such as pneumonia, streptococcal
infection, and typhoid fever. Our bodies have a special system
for combating the different infectious and toxic agents. This is
comprised of blood leukocytes (white blood cells) and tissue
cells derived from leukocytes. These cells work together in two
ways to prevent disease: by actually destroying invading
bacteria or viruses by phagocytosis, and by forming antibodies
and sensitized lymphocytes, one or both of which may destroy
or inactivate the invader.

Allocation of leucocytes in the organism

There are three locations of the leucocytes in the

organism: red bone marrow (30%), peripheral blood (20%) and
peripheral tissues (50%).
I Red bone marrow. There are 4 pools of leucocytes:
- pool of stem cells, which are in rest state. It is
insignificant and it is the reserve for blood formation;
- mitotic pool. These cells are in division state;
- maturating pool. These cells are differentiating; their
maturating period is 3-5 days;
- reserve pool. These are mature leucocytes, which can
enter the bloodstream.

88
 II Peripheral blood. There are 2 pools of leucocytes:
- pool of circulating leucocytes (50%);
- parietal (marginal) pool (50%).
III Peripheral tissues have:
- migrating leucocytes;
- leucocytes in rest state.

Functional properties of leucocytes

1 White blood cells, which have nucleus and other subcellular

structures.
2 External membrane is negatively charged.
3 They are able to produce amoeboid movement, so they can
move not only by the bloodstream, but also against the
bloodstream. White blood cells move through tissue spaces by
ameboid motion.
Both neutrophils and macrophages can move through the
tissues by ameboid motion. Some cells move at velocities as
great as 40 mm/min, a distance as great as their own length
each minute.
4 They are able to pass through the vessel wall out of the
bloodstream into the tissues. This ability is called diapedesis.
Leucocytes are circulating in the bloodstream for 4-72 hours,
than they stay in tissues. Blood is an intermediate medium for
the leucocytes existence.
White Blood Cells enter the tissue spaces by diapedesis.
Neutrophils and monocytes can squeeze through the pores of
the blood capillaries by diapedesis. That is, even though a pore
is much smaller than a cell, a small portion of the cell slides
through the pore at a time; the portion sliding through is
momentarily constricted to the size of the pore.

5 Chemotaxis. Many different chemical substances in the

tissues cause both neutrophils and macrophages to move

89
toward the source of the chemical. This phenomenon is known
as chemotaxis. When a tissue becomes inflamed, at least a
dozen different products are formed that can cause chemotaxis
toward the inflamed area. They include some of the bacterial or
viral toxins, degenerative products of the inflamed tissues
themselves, several reaction products of the “complement
complex” activated in inflamed tissues, and several reaction
products caused by plasma clotting in the inflamed area, as
well as other substances. The concentration is greatest near the
source, which directs the unidirectional movement of the white
cells. Chemotaxis is effective up to 100 micrometers away
from an inflamed tissue. Therefore, because almost no tissue
area is more than 50 micrometers away from a capillary, the
chemotactic signal can easily move hordes of white cells from
the capillaries into the inflamed area (fig. 4.1).

Figure 4.1 - Movement of neutrophils by diapedesis through capillary pores

and by chemotaxis toward an area of tissue damage.

90
 Movement of neutrophils by diapedesis through capillary
pores and by chemotaxis toward an area of tissue damage
6 They have high fermentative activity due to presence of
enzymes-hydrolases, polypeptidases, peroxidases, lipases.
7 They synthesize substances, which neutralize toxins.
8 They are able to absorb substances on their surface and
transport them.
9 They have phagocytic activity.
10 The term of life – few hours to few days (the shortest term –
granulocytes – minutes, hours, maximum 8-10 days; the
longest term – T-lymphocytes – months, years).

Life of white blood cells

Lifespan of white blood cells are not constant. It depends

upon the demand in the body and their function. Lifespan of
these ceils may be as short as half a day or it may be as long as
3-6 months. However, the normai lifespan of white biood cells
is as follows:
Neutrophils — 2-5 days
Eosinophils — 7-12 days
Basophils — 12-15 days
Monocytes — 2-5 days
Lymphocytes — 1/2-1 day
The amount in peripheral blood – 4-9 · 109/liter.
The decrease of leucocytes amount is called leucopenia, the
increase – leucocytosis.
There are 2 types of leucocytes:

I Physiological – is normal, physiological reaction of the

organism in some irritations. There are following types,
dependently on their causes:
1) emotional leucocytosis (occurs in result of emotional
stresses);

91
 2) myogenic (occurs in result of intensive physical
exercises);
3) static (occurs in result of change of the position of the
human body from horizontal to vertical);
4) alimental (occurs during or after eating);
5) painful (occurs during strong painful feelings);
6) leucocytosis of pregnant;
7) leucocytosis of newborn.

II Pathological (reactive) – it is connected with the

pathological process in the organism. Its reasons:
1) infectious diseases;
2) inflammatory processes;
3) allergic reactions;
4) intoxications of endo- and exogenous origin.

The difference between physiological

and reactive leucocytosis

Physiological leucocytosis:
1) it is redistributing (leucocytes from the parietal pool
are moving into circulation);
2) it has transient character (it is normalizing fast after
the cause disappears);
3) leukogram does not change (the correlation between
different forms persists);
4) degenerative forms of leucocytes do not appear.

Reactive leucocytosis is connected with the increase of

proliferation and maturating of leucocytes in red bone marrow
or increase of moving of reserve leucocytes from RBM to the
blood. During pathological leucocytosis the correlation
between different forms of leucocytes is disturbed.

92
 Percentage ratio between different forms of leucocytes is
called leukogram (formula of Arnet-Shilling).

Leukogram of a healthy adult human:

Leucocytes
Granulocytes Agranulocytes
eosinophils

neutrophils

lymphocytes

monocytes
basophils

immature stab segmented

0-1% 1-5% 0-1% 1-6 47-72% 19-37% 3-11%

Healthy human has instant leukogram and any changes in

it – is the signal to different sicknesses. The disturbance in
correlation between immature and mature forms of neutrophils
is called shift of leukogram. There is shift to the left and shift
to the right.
Shift to the left is characterized byincrease in the content
of immature neutrophils. Myelocytes appear in blood, the
amount of metamyelocytes increase. This happens during
leucocytosis.
Shift to the right is characterized with domination of
mature neutrophils with big amount of segment (5-6) on the
background of disappearance of immature forms. This testifies
the development of inflammatory process.
The correlation between mature and immature forms of
neutrophils is Bobrov’s index:

93
 monocytes immature  stab
BI   0,6  0,7
segmented

The important index is correlation between neutrophils

and lymphocytes.
Newborns have larger neutrophils content than
lymphocytes (Fig. 4.2). On 4-5 day the amount of neutrophils
decreases and the amount of lymphocytes increases. And till
the age of 5-6 the child has more lymphocytes than
neutrophils. In the age of 5-6 the amount of neutrophils
increases, and the amount of lymphocytes decreases. Starting
from this age the amount of neutrophils is higher than
lymphocytes.

4-5 day 5-6 years

Neutrophils

Lymphocytes
І decussation ІІ decussation

Figure 4.2 - Correlation of neutrophils and lymphocytes throughout life

94
Figure 4.3 – Types of leucosyses

95
 Main functions of leucocytes

Basophiles
Granules of these cells dye with basic stains into blue
color. Granules contain heparin, histamine, serotonin,
peroxidase, acid phosphatase, histidinecarboxylase (the
enzyme of histamine synthesis). Phagocyte activity is low.

Functions of basophiles
1 Participate in allergic reactions. Antigen-antibody complex
affects degranulation of basophiles and histamine release. In
low concentrations histamine interacts with H1-receptors and
causes basic manifestations of allergic reactions: dilatation
of blood vessels, increase of vessel wall permeability,
irritation of nervous endings, which cause itching, pains, and
increase of formation and secretion of mucus in respiratory
pathways, contraction of smooth muscles in bronchi. In big
concentrations histamine interacts with H2-receptors and
causes extinction of these reactions due to inhibition of
basophiles degranulation.
2 Participate the development of inflammation, especially
during last (regenerative) phase: heparin predicts blood
coagulation in inflammation source; histamine dilates
capillaries that help resolution and healing.
3 Regulation of vessel wall permeability (increased by
histamine and serotonin).
4 Participates hemostasis (heparin is an anticoagulant,
histamine causes vessel spasm after damage).
The increase of basophiles amount (basophilia) is rare. It
can cause the development of chronic myeloleucosis,
hemophilia, and polycythemia.

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 Eosinophils
Granules of these cells dye with acid stains into pink
color. They have phagocyte activity, but due to low eosinophil
concentration in blood their role in this process is insignificant.
The concentration of eosinophils in blood is variable during
daytime, which is defined by hydrocortisone level (the
maximum quantity of eosinophils is at night, the minimum
quantity – in the morning). They stay in blood for 3-8 hours,
then they migrate into the connective tissue, where they
perform their functions. They contain granules of two types.
Granules of 1st type have protein, which has arginine;
hydrolytic enzymes; peroxidases; histaminases; esterases.
Granules of 2nd type have acid phosphatase and arylsulfatase.

Functions of eosinophils
1 Taking part in allergic reactions: histaminase splits
histamine, which results in termination of allergic reactions;
arylsulfatase degrade anaphilaxin; they can synthesize and
release factor that inhibits the release of histamine from
basophils.
2 Degradation of toxins with protein nature.
3 Taking part in neutralization of toxins which are made by
parasites (helminthes).
4 Taking part in fibrinolysis (production of plasminogen).
5 Delaying the spread of inflammation, decrease the
performance of inflammation process (due to histamine
neutralization).
The increase of eosinophils amount (eosinophilia) can be
observed when allergic reactions, bronchial asthma,
helminthosis, chronic myeloleucosis, some infantile infections
(scarlet fever), after taking some kind of drugs (antibiotics,
sulfanilamides).

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 Neutrophils
Granules of these cells can be dyed with acid or basic
stains into pink-violet color. Only 1% out of all neutrophils is
circulating in the bloodstream, others are in tissues. They
contain 2 types of granules. Primary granules have hydrolytic
enzymes (acid phosphatase, β-glucuronidase, acid protease,
arylsulfatase); myeloperoxidase, lizocim.
Secondary granules contain basic phosphatase, main
cationic proteins, phagocytins, lactoferin, lizocim,
aminopeptidase.
In dependence of age they have different shape of nucleus. In
dependence of nucleus shape there are immature, stab and
segmented neutrophils. They circulate in blood for 4-8 hours,
and then they move into tissues, where they live 4-5 days.

Functions of neutrophils
1 Phagocytosis. Neutrophils are important elements of non-
specific protection of an organism. They are the first to
come to infection source or damage spot. Neutrophils
neutralize not self agents with the help of their enzymes.
Neutrophils can die there. Dead neutrophils form pus.
For the characteristic of phagocytic activity of
neutrophils the following indexes are used:
 The percent of cells which work as phagocytes (normally
– 68,5 - 99,3%).
 Phagocytic index (the number of agents, which 1 cell can
consume, normally – 12 - 23);
2 Secretion of germicide substances (lysosomal cationic
proteins, histones, lactoferin).
3 Antiviral activity (production of interferon).
4 Stimulation of tissue regeneration after damage (synthesis of
acid glycosaminoglycans).

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 5 Participate in specific immunity by affecting the
activity of T- and B-lymphocytes, increasing the
amount of antibodies.
6 Secretion of substances that dilate blood vessels.
7 Sexualization of the blood. Most of neutrophils of
women have satellites of nucleus that are surrounding
the nucleus. One of X-chromosome is located there.
That is why they are called sex chromatin (Bar’s
bodies). Presence or absence of these satellites allows
defining possible sexualization of the blood.

The mechanism of phagocytosis

Phagocytosis – is active devourment of the solid
substances by cells. Cells, which are capable of phagocytosis,
are called phagocytes. There are poly phagocytes (neutrophils)
and mononuclear phagotyces (monocytes).
Phagocytes must be selective of the material that is
phagocytized; otherwise, normal cells and structures of the
body might be ingested. Whether phagocytosis will occur,
depends especially on three selective procedures. Firstly, most
natural structures in the tissues have smooth surfaces, which
resist phagocytosis. But if the surface is rough, the likelihood
of phagocytosis is increased. Secondly, most natural substances
of the body have protective protein coats that repel the
phagocytes. Conversely, most dead tissues and foreign
particles have no protective coats, which make them subject to
phagocytosis. Thirdly, the immune system of the body
develops antibodies against infectious agents such as bacteria.
The antibodies then adhere to the bacterial membranes and
thereby make the bacteria especially susceptible to
phagocytosis. To do this, the antibody molecule also combines
with the C3 product of the complement cascade, which is an
additional part of the immune system discussed in the next
chapter. The C3 molecules, in turn, attach to receptors on the

99
 phagocytic membrane, thus initiating phagocytosis. This
selection and phagocytosis process is called opsonization.

Stages of phagocytosis:
I Conjugation stage. Phagocyte moves to direction of not self
agent (chemotaxis).
II Adhesion stage. Phagocyte interacts with the agent. There
are two mechanisms:
1) without receptor: electrostatic and hydrophobic interaction
(phagocyte is negatively charged, positive particles);
2) with receptor. On the surface of macrophages there are
receptors for opsonin-substances that can interact with
bacteria.
III Devourment stage. Its steps:
 invagination of phagocyte membrane on the contact place;
 the formation of phagosome, which contains the agent;
 the formation of phagolysosome: consolidation of
phagosome with lysosomes (secondary granules).
IV Digestive stage. Its steps:
 The disposal of bacteria – intercellular cytolysis with the
help of germicide systems of phagocytes (myeloperoxidase
system, which produces hypochloride ion ClO -, free
radicals and peroxides O30, HO20, OH0, lisocim, lactoferin,
non-enzymatic cationic proteins, lactic acid).
 Digestion – hydrolysis of killed bacteria with the help of
hydrolytic enzymes.

Monocytes
Monocytes are the largest blood cells, which does not
have granules. They secrete more than 100 biologically active
substances. Monocytes have the highest phagocytic activity
among all blood cells. Monocytes are formed in RBM and they
enter the bloodstream when they are not mature. Monocytes
stay in blood for 2-3 days, after that they move into tissues. In

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tissues monocytes are growing, the amount of lysosomes and
mitochondria. After maturating, monocytes turn into immobile
cells histiocytes (tissue macrophages).

Functions of monocytes
1 Participation in the development of inflammatory process.
Monocytes appear in the inflammation site after neutrophils.
They perform phagocytosis in acid medium, where neutrophils
loose their activity. In inflammation site monocytes englobe
germs, dead leucocytes, damaged cells. They clean the
inflammation site and prepare it for regeneration. Monocytes
are called “organism cleaners” for performing this function.
2 Participating in the process of regeneration. Monocytes
release factors, which stimulate growth of endothelial and
smooth-muscles cells, also they release fibrinogenic factor,
which increases the rate of collagen synthesis.
3 Formation of “protection wall” around not-self bodies,
which can not be destroyed by enzymes.
4 Participation in formation of specific immunity. Monocytes
englobe, transform and present antigen to immunocompetent
cells (T and B-lymphocytes); they participate cooperation of T
and B-lymphocytes.
5 Antitumoral and antiviral action, which is provided by the
secretion of lizocim, interferons, elastase, collagenase.
6 Participation in the development of fever. They release
endogenic leucocytory pyrogen (interleukin-1), which affects
the thermoregulation center and causes the increase of body
temperature.
7 Participating in the process of complement formation.
8 Participating in blood formation. Monocytes form
interleukins, which affects leukopoesis.
9 Participating in metabolism of lipids and iron.

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 The definition of mononuclear
phagocytory system (MPS)
MPS includes:
 osteoclasts;
 monocytes;
 macrophages of connective tissue;
 macrophages of liver and spleen;
 macrophages of RBM;
 macrophages of the lungs;
 microgliocytes.
The main features of cells of this system are:
1) phagocyte activity;
2) the presence of receptors for antibodies and
complement;
3) common origin and morphology.

Reticuloendothelial system (RES)

RES consists of tissue macrophages (fixed monocytes)
which are present in lymph nodes, spleen, Liver, bone marrow
and connective tissue.

Functions of RES:
1) Defence: The defensive mechanism include:
a) Formation of antibodies against the invading agents
(immune response).
b) Phagocytosis and digestion of bacteria and protozoa.
c) Engulfing foreign particles, e.g. dust and carbon.
2) Rapair: of tissue after inflammation by removal of dead
tissue and provide protein and fat needed for repair.
3) Blood formation: reticuloendothelial cells (RECs) of bone
marrow and spleen may change to haemocytoblasts to form
blood cells.

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4) Removal of old blood cells: from circulation and formation
of bile pigments.
5) Storage of iron: needed for erythropoiesis.

Spleen: It is the largest lymphoid organ.

Functions:
1) defence:
a) phagocytosis: by the RECs in the spleen;
b) antibody formation: lymphocytes and plasma
cells (immune response).
2) blood cell formation:
a) erythropoiesis: In the middle 1/3 of intra-uterine
life. If adult bone is diseased or destroyed,
reticuloendothelial cells in the spleen is changed to
haemocytoblasts to form blood cells (extramedullary
haemopoiesis);
b) lymphopoiesis: Splenic lymph nodes form
lymphocytes and plasma cells. They play an important
role in immune response;
c) iron storage needed for erythropoiesis.
3) removal of old cells:
a) old RBCs, leucocytes and platelets are removed
by RECs of spleen;
b) Hb breakdown and bile pigment formation.
Hypersplenism: is a congenital condition in which spleen
engulf-the normal blood cells leading to anaemia and
thrombocytopenic purpura.
4) Blood Reservoir:
In animals (dog & cat), the blood sinuses can
accomodate 1/4 of the blood volume, but much less in
man (about 200 ml). The stored blood is squeezed out of
spleen to general circulation by contraction of the plain

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muscle fibres of splenic capsule (supplied by sympathetic
nerves) in response to:
 O 2 lack which stimulates the sympathetic nerves.
 Haemorrhage.
 Muscular exercise, hot climate, emotions or adrenaline
release.
Lymph nodes:
They lie between lymph vessels  efferent lymph
vessels  finally into thoracic duct which opens into the
junction of the left subclavian with internal jugular veins
venous circulation.

Macrophages in the Lymph Nodes

Essentially no particulate matter that enters the

tissues, such as bacteria, can be absorbed directly
through the capillary membranes into the blood. Instead,
if the particles are not destroyed locally in the tissues,
they enter the lymph and flow to the lymph nodes located
intermittently along the course of the lymph flow. The
foreign particles are then trapped in these nodes in a
meshwork of sinuses lined by tissue macrophages.
Function of lymph nodes
1 Filtration: Removal of foreign particles e.g. carbon
particles.
2 Removal of bacteria: The lymph nodes enlarge
(macrophages increase mucn in number). They are the
defence mechanism against local infection.
3 Formation of lymphocytes and plasma cells: which
leave the nodes through the efferent lymph vessels to
reach the blood stream.
4 Production of antibodies: by lymphocytes as a defence

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mechanism.
Liver:

Bacteria pass through gastrointestinal mucosa

portal blood  phagocytosis by kupffer cells lining blood
sinuses of the liver and no I bacteria pass to systemic
circulation.

Macrophages (Kupffer Cells)

in the Liver Sinusoids
Still another favorite route by which bacteria
invade the body is through the gastrointestinal tract.
Large numbers of bacteria from ingested food constantly
pass through the gastrointestinal mucosa into the portal
blood. Before this blood enters the general circulation, it
passes through the sinusoids of the liver; these sinusoids
are lined with tissue macrophages called Kupffer cells,
shown in Figure 4.5. These cells form such an effective
particulate filtration system that almost none of the
bacteria from the gastrointestinal tract succeeds in
passing from the portal blood into the general systemic
circulation. Indeed, motion pictures of phagocytosis by
Kupffer cells have demonstrated phagocytosis of a single
bacterium in less than 1/100 of a second.

105
Figure 4.5 – Kupffer cells lining the liver sinusoids, showing phagocytosis
of India ink particles into the cytoplasm of the Kupffer cells.
Lymphocytes
Unlike other leukocytes, lymphocytes live not for few
days, but for few years, some of them live throughout human’s
life.
Role of lymphocytes in the organism
1 They are the central element of immune system; they are
responsible for the formation of non-specific immunity.
2 They perform the role of “censorship” in the organism: they
provide the protection of every non-self agent; provide genetic
constancy of the internal medium.
3 They provide rejection of transplants reaction.
4 They neutralize own mutated cells.

There are 3 types of lymphocytes:

1) Thymus-dependent – T-lymphocytes (40-70%);
2) Bursa-dependent – B-lymphocytes (20-30%);
3) Zero – O-lymphocytes (10-20%).

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 Lymphocytes are formed in RBM; they differentiate in
primary lymphatic organs: T-lymphocytes – in thymus, B-
lymphocytes – in bone marrow. Then lymphocytes enter the
blood and stay in secondary lymphatic organs: lymphatic
nodules, spleen, lymphatic tissue of gastrointestinal tract;
respiratory pathways, where proliferation of lymphocytes
occurs as an answer to the intruding not-self antigen into the
organism.

The main function of T-lymphocytes

is cellular immune reactions
There are different forms of T-lymphocytes:
1 T-killers. These lymphocytes neutralize cells that carry
antigen. One killer can neutralize one antigen-carrying cell. T-
killers are able to destroy tumor cells, cells of nonself
transplants, mutant-cells. They can form mediators of
immunity – lymphokines.
2 T-helpers. These cells distinguish antigen, interact with B-
lymphocytes and help them to turn into plasmatic cells.
Helpers’ product interleukin-2, which helps differentiation of
additional T-cells and also the factor of B-cells growth that
helps differentiation of B-lymphocytes into plasmatic cells.
3 T-suppressors. These cells suppress overabundant activity of
T- and B-lymphocytes, formation of antibodies, predicting in
this way excess immune response. T-suppressors provide
formation of immune tolerance (lack of immune response to
self antigens and for the one, the organism already had contact
with).
4 T-amplifiers. These cells activate T-killers. They regulate
correlation between killers and suppressors.
5 T-memory cells. These cells circulate in blood for years and
after repeated contact, they distinguish antigen and provide
secondary immune response, which is faster and more
intensive.

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 The main function of B-lymphocytes is providing
humoral immune response by synthesis of antibodies. After
contacting antigen B-lymphocytes migrate into secondary
lymphatic organs, there they multiply and transform into
plasmatic cells that are able to produce 5 types of
immunoglobulins: IgM, IgG, IgE, IgA, and IgD.
There few forms of B-lymphocytes.
1 B1 lymphocytes – are antecessors of antibody-forming cells.
2 B2 lymphocytes. These are B-suppressors. They suppress
development and transformation of T- and B-lymphocytes
into effector cells.
3 B3 lymphocytes. These are B-killers, which have cytotoxic
activity.
O-lymphocytes do not pass through differentiation and in case
of necessity can transform into T- and B-lymphocytes. K-
cells (killer-cells) and natural killer cells (NKC) also belong
to O-lymphocytes; they are responsible for non-specific
resistance and are especially active against tumor cells.

Regulation of formation and activity of leucocytes

Regulation of leucocytes formation is performed by
leucopoietins and inhibitors of leucopoiesis. Stimulators of
leucopoiesis are leucopoietins. The main source of
leucopoietins is activated macrophages. Interleukin-1, factor of
tumoral necrosis, colony-stimulating factor belong to
leucopoietins. Colony-stimulating factor is the most learned
which stimulates the formation of granulocytes in RBM.
Interleukin-1 increases transport of reserve leucocytes from
RBM into the bloodstream.
Inhibitors of leucopoiesis are high-molecular inhibitors
of blood serum – lipoprotein, lactoferin, and keylons.
Migration of leucocytes into tissues is regulated by local

108
factors-chemotaxins. Interaction between leucocytes is
regulated by cytokins.
There are two types of cytokins
1 Lymphokins. They are formed in lymphocytes. These are
cytotoxins, chemotaxins, and mitogens.
2 Monokins. They are formed in monocytes, macrophages.
These are interleukin-1, factor of tumoral necrosis, colony-
stimulating factor.

109
 Questions for self-control

1 Structure of the protective system of the organism.

2 Location of leucocytes in the organism.
3 Functional properties of leucocytes.
4 Definition of leucopenia and leucocytosis. Types of
leucocytosis.
5 Leukogram. Definition of leukogram shift to the right and to
the left. Definition of leukogram decussation.
6 Main functions of leucocytes.
7 Mechanism of phagocytosis.
8 Definition of mononuclear phagocytic system.
9 Regulation of formation and activity of leucocytes.

Tests for self-control

1 Which blood cells are leucocytes:

a) monocytes;
b) lymphocytes;
c) thrombocytes;
d) eosinophils;
e) basophils;
f) erythrocytes
g) neutrophils?

2 Which cells provide specific (immune) protection from

nonself agents:
a) monocytes;
b) lymphocytes;
c) thrombocytes;
d) eosinophils;
e) basophils;

110
 f) erythocytes
g) neutrophils;
h) no correct answer?

3 Which blood cells are granulocytes:

a) monocytes;
b) lymphocytes;
c) thrombocytes;
d) eosinophils;
e) basophils;
f) erythrocytes;
g) neutrophils;
h) no correct answer?

4 Which blood cells are capable for phagocytosis:

a) monocytes;
b) thrombocytes;
c) B-lymphocytes;
d) T-lymphocytes;
e) erythrocytes;
f) neutrophils;
g) no correct answer?

5 What are functions of neutrophils:

a) transport of oxygen;
b) participating blood stoppage;
c) providing specific (immune) protection;
d) phagocytosis;
e) albumin synthesis of blood plasma;
f) no correct answer?

6 What are the functions of T-lymphocytes:

a) transport of oxygen;
b) participating blood stoppage;

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 c) providing specific (immune) protection;
d) phagocytosis;
e) albumin synthesis of blood plasma;
f) no correct answer?

7 Which of cells provide immunoglobulin synthesis:

a) B-lymphocytes;
b) T-lymphocytes;
c) O-lymphocytes;
d) macrophages;
e) neutrophils;
f) eosinophils;
g) basophils;
h) no correct answer?

8 What is leukogram:
a) percent of leucocytes among all formed blood elements;
b) absolute content of particular forms of leucocytes in unit
of volume;
c) percent of mature form of leucocytes among their
antecessors;
d) percent correlation between particular forms of
leucocytes in peripheral blood;
e) no correct answer?

9 Which fraction of blood plasma proteins do antibodies

belong to:
a) albumins;
b) α1-globulins;
c) α2-globulins;
d) β-globulins;
e) γ-globulins;
f) no correct answer?

112
10 What are the functions of B-lymphocytes:
a) transport of oxygen;
b) participating in blood stoppage;
c) providing specific (immune) protection;
d) phagocytosis;
e) albumin synthesis by blood plasma;
f) no correct answer?

113
CHAPTER 5 Haemostasis

Haemostasis system is the system, which provides

maintenance of the blood fluidity and stoppage of bleeding
after injure of blood vessels.

Functions of haemostasis system

1 Provides maintenance of the blood fluidity.
2 Stops the blood when vessel wall is damaged.

Structure of haemostasis system

1 Blood vessels walls.
2 Formed blood elements.
3 Biochemical systems of blood plasma:
 system of blood coagulation;
 anticoagulative system;
 fibrinolytic system;
 calicrein-kinine system.

Mechanisms of haemostasis
1 Vessel-thrombocytory (primary, microcirculatory).
Provides stoppage of bleeding in vessels of
microcirculatory system with diameter less than 100 mkm.
Vessel wall and thrombocytes take part in this mechanism.
Results in white clot, which consists of thrombocytes.
2 Coagulatory (secondary, macrocirculatory). It is the
continuation of vessel-thrombocytory and it is based on it.
Provides stoppage of bleeding in vessels with diameter more
than 100 mkm. Results in red clot, which consists of fibrin and
formed blood elements.

The role of vessel wall in haemostasis

I Vessel wall participate activation of haemostasis with the
help of following mechanisms:

114
 1) Unmasking of the collagen
When blood vessel wall is damaged the collagen is
unmasked. So it becomes available for the formed blood
elements. Collagen provides contact activation of thrombocytes
and Hageman’s factor (F. XII), which initiates the internal
mechanism of blood coagulation.
2) Release of ADP
ADP is released from the damaged cells of vessel wall,
and it is powerful activator of thrombocytes adhesion and
aggregation.
3) Release of tissue thromboplastin
Thromboplastin is released from damaged cells of vessel
wall and initiates external mechanism of blood clotting and
formation of small amount of thromboplastin in the damaged
place.
4) Release of Willebrant factor
Endotheliocytes of vessel wall form Willebrant factor –
glycoprotein, which participates thrombocytes adhesion.

II Vessel wall provides maintenance of blood fluidity

(thromboresistancy) with the help of following mechanisms:
1) Formation of prostacyclin. Prostacyclin is formed by
endotheliocytes (derivative of arachidonic acid), which is
powerful inhibitor of thrombocytes aggregation.
2) Formation of antithrombin III – powerful natural
anticoagulant.
3) Ability of endothelium to fix heparin-antithrombin III
complex on its surface, which increases activity of this
complex in hundreds times.
4) Formation of fibrinolysis activators.
5) Endothelial Surface Factors. Probably the most
important factors for preventing clotting in the normal
vascular system are the smoothness of the endothelial cell
surface, which prevents contact activation of the intrinsic

115
 clotting system; a layer of glycocalyx on the endothelium
(glycocalyx is a mucopolysaccharide adsorbed to the
surfaces of the endothelial cells), which repels clotting
factors and platelets, thereby preventing activation of
clotting; and a protein bound with the endothelial
membrane, thrombomodulin, which binds thrombin. Not
only does the binding of thrombin with thrombomodulin
slow the clotting process by removing thrombin, but the
thrombomodulin- thrombin complex also activates a
plasma protein, protein C, that acts as an anticoagulant
by inactivating activated Factors V and VIII.

Role of thrombocytes in haemostasis

Thrombocytes of the peripheral blood are the fragments

of cells – megacariocytes, which in bone marrow break down
into 3-4 thousands of small parts of blood platelets.
Thrombocyte does not have nucleus and most of
subcellular structures. At the same time it has complicated
structure and well adapted to the functions that it should
perform. There are a lot of biologically active substances on
the membrane and inside thrombocyte: partially formed by
thrombocyte itself and part of them get into it from the blood
plasma. Most of substances are in granules. There are 4 types
of granules:
1st type: include such non-protein structures as ATP, ADP,
serotonin, pyrophosphate, adrenalin, calcium.
2nd type: include low-molecular proteins, Willebrant factor,
fibrinogen.
3rd and 4th type: include enzymes.
In their cytoplasm are such active factors as actin and myosin
molecules, which are contractile proteins similar to those found
in muscle cells, and still another contractile protein,
thrombosthenin, that can cause the platelets to contract;

116
residuals of both the endoplasmic reticulum and the Golgi
apparatus that synthesize various enzymes and especially store
large quantities of calcium ions; mitochondria and enzyme
systems that are capable of forming adenosine triphosphate
(ATP) and adenosine diphosphate (ADP); enzyme systems that
synthesize prostaglandins, which are local hormones that cause
many vascular and other local tissue reactions; an important
protein called fibrin-stabilizing factor,which we discuss later in
relation to blood coagulation; and a growth factor that causes
vascular endothelial cells, vascular smooth muscle cells, and
fibroblasts to multiply and grow, thus causing cellular growth
that eventually helps repair damaged vascular walls. The cell
membrane of the platelets is also important.
On its surface is a coat of glycoproteins that repulses adherence
to normal endothelium and yet causes adherence to injured
areas of the vessel wall, especially to injured endothelial cells
and even more so to any exposed collagen from deep within
the vessel wall. In addition, the platelet membrane contains
large amounts of phospholipids that activate multiple stages in
the blood-clotting process, as we discuss later.
Thrombocytes live for 8-12 days. They are degraded in liver,
spleen, lungs or adhere to endothelium of vessels and perform
trophic function.
Normally there are 180-320•109/liter of thrombocytes in blood.
The decrease of thrombocytes amount is called
thrombocytopenia, the increase is thrombocytosis. There are
daily changes of thrombocytes amount: the amount is higher at
day than at night. Their amount changes after physical
exercises, after eating and after stress.

Functions of thrombocytes
1 Angiotrophic function. Daily 10-15% of all thrombocytes
that circulate in blood are used as a nourishment supply of the
vessel wall. They adhere to the endothelium, degrade and their

117
content outpour on endothelium. The main component of this
content is thrombocytory growth factor, which harden vessel
wall, especially the wall of capillaries.
If endothelial cells lose their endothelial nourishment
(occurs when thrombopenia), they become dystrophic and
erythrocytes are able to pass through it. Diapedesis of
erythrocytes can be very intensive. Externally it shows up
as small hemorrhages – petechiae.
2 Transport. Thrombocytes are able to absorb biologically
active substances and transport them.
3 Participating blood clotting. There are a number of
substances in thrombocytes, which participate blood clotting.
These are thrombocytory (platelet) factors. The most important
are:
a) Factor 3 (thrombocytory thromboplastin). It is
phospholipids, which is released after thrombocytes
degradation and it is used as matrix for the reactions for
the first phase of clotting.
b) Factor 4 (antiheparin factor) – binds heparin, increases
coagulation rate.
c) Factor 5 (fibrinogen) provides compression and
contraction of blood clot.
d) Factor 6 (thrombostenin) protein, which is like
actomiosin of skeletal muscles has ATPase activity.
e) Factor 10 (pressor factor) – serotonin, which is absorbed
by thrombocytes in blood.
f) Factor 11 (aggregation factor) – ADP and thromboxan A 2
that provide aggregation.
4 Participating stoppage of bleeding. It is determined by the
ability of thrombocytes to adhesion and aggregation, which
leads to the formation of thrombocytory cork.

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 Vessel-thrombocytory haemostasis

Vessel-thrombocytory haemostasis is performed in several

stages:
 spasm of arteries;
 adhesion of thrombocytes;
 aggregation of thrombocytes;
 release reaction;
 thrombus consolidation.

I Spasm of arteries is the contraction of vessels when they

are damaged
There are two types of vessel spasm:
1 Primary. It has reflex origin. Appears in first seconds of
damage with the help of sympathetic nervous system.
2 Secondary. Due to release of biologically active substances
in the damage spot (serotonin, adrenalin, thromboxan).
Appears in few seconds after damage.
Spasm of vessels develops fast enough and disappears in
few seconds and bleeding continues.
The nervous reflexes are initiated by pain nerve impulses
or other sensory impulses that originate from the traumatized
vessel or nearby tissues. However, even more vasoconstriction
probably results from local myogenic contraction of the blood
vessels initiated by direct damage to the vascular wall. And, for
the smaller vessels, the platelets are responsible for much of
the vasoconstriction by releasing a vasoconstrictor substance,
thromboxane A2. The more severely a vessel is traumatized,
the greater the degree of vascular spasm. The spasm can last
for many minutes or even hours, during which time the
processes of platelet plugging and blood coagulation can take
place.
II Adhesion of thrombocytes – is the thrombocytes to
the damaged vessel wall.

119
 The main reason of adhesion is unmasking of
collagen.
There are 2 stages of adhesion:
1) Precontact stage. It is associated with the
changes in shape of thrombocytes. They take spheroid
shape with 3 to 10 processes.
2) Contact stage. It is associated with the
attachment of thrombocytes to the endothelium of blood
vessels. Thrombocyte can interact with vessel wall directly
and also with the help of special protein – Willebrant
factor.
Adhesion occurs easier with the help of two factors:
1) Reversion of the membrane charge after damage that
provides electrostatic co-operation of thrombocytes with
vessel wall.
2) Slow down of the blood movement in microcirculatory
vessels.
III Aggregation of thrombocytes – is the aggregation of
thrombocytes in the damage spot and conglutination of them
one to another.
The reasons of aggregation are aggregants.
Aggregants can have thrombocytory origin (those that are
released by thrombocytes) and not-thrombocytory (those that
are released by other cells or are formed in plasma). The main
aggregants are:
1) ADP;
2) Thromboxan A2 and arachidonic acid;
3) Biogenic amines (adrenalin, serotonin);
4) Factor of thrombocytes aggregation;
5) Thrombin;
6) Thrombospondin.

Stages of aggregation

120
1 Initial aggregation. It is performed at the same time with
adhesion. The main reason of it is ADP with not-
thrombocytory origin, which is released from damaged
cells.
2 Reverse aggregation. Aggregation, which can be stopped.
The main reason of it is thrombocytory ADP, thromboxan
A2, arachidonic acid.
3 Irreversible aggregation. Aggregation with the damage of
thrombocytes and it cannot be stopped. The reason of it is
thrombin.

Mechanism of aggregation
Aggregation is performed in two stages:
Stage 1 – stage of thrombocytes activation (fig. 5.1).
Aggregants increase thrombocyte membrane
permeability to calcium. By the concentration gradient calcium
enters thrombocytes. Its concentration increases in these cells.
Calcium causes following effects in thrombocytes:
1) contraction of myofibrils, which leads to formation of
processes;
2) increase of hydrolysis rate of ATP with the formation of
ADP, which is aggregant;
3) release (secretion) of granules;
4) activation of phospholipase A2, which leads to formation
of arachidonic acid and thromboxan A2.

121
 Pathogenic Biogenic Factors Other
factors amines damage of vessel aggregants

[Са2+]

myofibrils increase of release activation

contraction ATP hydrolysis of granules of phospholipase A2
Figure 5.1 – Mechanism of aggregations

Stage 2 – stage of thrombocytes conglutination.

There are two mechanisms of this stage:
1) formation of bridges between thrombocytes, which are
formed with Ca2+ and ADP;
2) formation of bridges between thrombocytes, which are
formed with plasma proteins. These proteins are
aggregation co-factors of plasma. Fibrinogen, albumins,
agrexons A and B.
IV Release reaction – is the process of granules secretion
in thrombocytes.
There 2 types of release reaction:
1) reaction of early release, which is performed during
adhesion and initial aggregation. Granules of 1 and 2 type
are released during this reaction;

122
 2) reaction of late release, which is performed during
irreversible aggregation. Granules of 3 and 4 type are
released during this reaction.
V Thrombus consolidation – packing, retraction of
thrombus in consequence of which it loses extra water and
become hard. Contraction of thrombus is performed with the
help of protein thrombostenin.

The scheme of vessel-thrombocytory haemostasis

Vessel wall damage Spasm of arteries

Adhesion and initial

Reaction of early
aggregation of
release
thrombocytes

Reverse aggregation

Irreversible Reaction of late

aggregation release

Thrombus
consolidation

Coagulatory haemostasis

Basic Theory More than 50 important substances that

cause or affect blood coagulation have been found in the blood
and in the tissues—some that promote coagulation, called
procoagulants, and others that inhibit coagulation, called

123
anticoagulants.Whether blood will coagulate depends on the
balance between these two groups of substances. In the blood
stream, the anticoagulants normally predominate, so that the
blood does not coagulate while it is circulating in the blood
vessels. But when a vessel is ruptured, procoagulants from the
area of tissue damage become “activated” and override the
anticoagulants, and then a clot does develop.
It is the cascade of biochemical reactions, which result in the
formation of fibrin. Plasma factors of coagulation participate
coagulative haemostasis:
 I – Fibrinogen;
 II – Prothrombin;
 III – Tissue thromboplastin. (tissue factor);
 IV – Ca2+ ions;
 V – Proaccelerin, Ac-globulin, labile factor;
 VI – Active form of factor V (accelerin);
 VII – Proconvertin, stable factor;
 VIII – Antihaemophilic globulin, antihaemophilic
factor A;
 IX – Christmas’s factor, antihaemophilic factor B;
 X – Stuart-Prauer’s factor, prothrombinase;
 XI – Plasma antecessor of thromboplastin,
antiheamophilic factor C;
 XII – Hageman’s factor, contact factor;
 XIII – Fibrinstabilizing factor, fibrinase.
All factors of coagulation can be divided into few groups:
a) substrate for the reaction – F. I
(fibrinogen);
b) Ca2+ ions;
c) accelerants of reactions: F. V
(proaccelerin), F. VIII (antihaemophilic globulin);
d) proteolytic enzymes: F. II, F. III, F. VII,
F. IX, F. X, F. XI, F. XII.

124
 Coagulative reactions are based on the reactions of
hydrolysis, which are performed by proteolytic enzymes.
Reactions occur in phospholipids of membranes of destroyed
erythrocytes and thrombocytes. Factors of coagulation are
fixed on membrane with the help of Ca2+ ions.
The main stages of blood coagulation are described by Moravic
in 1905.
There are 3 stages of blood coagulation:
Stage 1 – formation of active prothrombinase.
In response to rupture of the vessel or damage to the blood
itself, a complex cascade of chemical reactions occurs in the
blood involving more than a dozen blood coagulation factors.
The net result is formation of a complex of activated
substances collectively called prothrombin activator.
Stage 2 – formation of thrombin.
The prothrombin activator catalyzes conversion of
prothrombin into thrombin.
Stage 3 – formation of fibrin.
The thrombin acts as an enzyme to convert fibrinogen into
fibrin fibers that enmesh platelets, blood cells, and plasma to
form the clot itself.

Formation of prothrombinase
Prothrombin and Thrombin Prothrombin is a plasma
protein, an alpha2-globulin, having a molecular weight of
68,700. It is present in normal plasma in a concentration of
about 15 mg/dl. It is an unstable protein that can split easily
into smaller compounds, one of which is thrombin, which has a
molecular weight of 33,700, almost exactly one half that of
prothrombin. Prothrombin is formed continually by the liver,
and it is continually being used throughout the body for blood
clotting. If the liver fails to produce prothrombin, in a day or so
prothrombin concentration in the plasma falls too low to
provide normal blood coagulation. Vitamin K is required by

125
the liver for normal formation of prothrombin as well as for
formation of a few other clotting factors. Therefore, either lack
of vitamin K or the presence of liver disease that prevents
normal prothrombin formation can decrease the prothrombin
level so low that a bleeding tendency results.

Active prothrombinase is formed with the help of two

mechanisms:
1) extrinsic that is initiated by phospholipids, which are
released from damaged cells of vessels or from
connective tissue;
2) intrinsic that is initiated by coagulative factors of blood.
Extrinsic (tissue) mechanism – is fast mechanism, last for
5-20 seconds. Reactions occur on membranes of damaged
cells. After damage, cells (mainly lysosomes) excrete active
enzymes – tissue thromboplastin (F. IIIa) activates
proconvertin (F. VII). F.VIIa with phospholipids of tissues and
calcium form complex, which activate prothrombinase (F. X).
F. Xa with phospholipids, calcium and F. Va (proaccelerin)
form complex, which is tissue prothrombinase.

Damage
to cells

F. ІІІ

F. VІІ F. VІІа

F. Х F. Ха

Са2+
Са2+

126
 Membranes of damaged cells

Tissue prothrombinase activates small amount of

thrombin, which:
1) is used for the thrombocytes aggregation;
2) provides formation of receptors on membranes of
aggregated thrombocytes that bind F. Xa, resulting in its
unavailability for anticoagulants, mainly for antithrombin
III.
Intrinsic (blood) mechanism – is durable, last for 5-7
minutes. Reactions occur on the membranes of damaged blood
cells (thrombocytes, erythrocytes). Collagen fibers initiate the
process, they uncover after damage of vessel. Contact of
collagen with F. XII (Hageman’s factor, contact), provides
activation of F. XII. F. XIIa, F. XIa and phospholipids form
complex, which activate F. X. F. Xa together with Ca 2+ ions
and F. Va also form complex, which is blood prothrombinase.

Contact or fermentative activation

F. ХІІ F .ХІІа

F. ХІ F. ХІа

F. ІХ F. ІХа + F.
VІІІ
Са2+
F. Х F. Ха

Са2+ Са2+
Са2+
Factor III of thrombocytes Са2+

127
 In pathological case there is third mechanism of
prothrombinase formation – macrophagal. Endotoxins of
bacteria, immune complexes, complement, products of tissues
degeneration act on macrophages, provide release of active
prothrombinase from them (F. Xa). This mechanism has
adaptive role, because spread of pathogenic factors in the
organism is limited by the blood coagulation.
Internal and external mechanisms are associated with
each other with the help of calicrein-kinine system.

F. XІІ, ХІ, ІХ,

F. ІІІ, VІІ Calicrein-
kinine VІІІ
Phospholipids system
Factor 3 of
of cell
thrombocytes
membranes

So, the result of the first stage is formation of tissue and

blood prothrombinase.
Formation of thrombin
This is fast stage (2-5 sec). Blood prothrombinase absorb
prothrombin on its surface and in presence of calcium
transform it to thrombin.

Prothrombinase + F. V

prothrombin (F. ІІ) thrombin

Са2+

Са2+ Са2+ Са2+

Phoespholipids of cell membranes,

F. 3 of thrombocytes

128
 Thrombin is a protein enzyme with weak proteolytic
capabilities. It acts on fibrinogen to remove four low-
molecularweight peptides from each molecule of fibrinogen,
forming one molecule of fibrin monomer that has the automatic
capability to polymerize with other fibrin monomer molecules
to form fibrin fibers.
So, the result of the second stage is formation of
thrombin.
Formation of fibrin

Fibrinogen is a high-molecular-weight protein (MW =

340,000) that occurs in the plasma in quantities of 100 to 700
mg/dl. Fibrinogen is formed in the liver, and liver disease can
decrease the concentration of circulating fibrinogen, as it does
the concentration of prothrombin, pointed out above.
Because of its large molecular size, little fibrinogen
normally leaks from the blood vessels into the interstitial
fluids, and because fibrinogen is one of the essential factors in
the coagulation process, interstitial fluids ordinarily do not
coagulate. Yet, when the permeability of the capillaries
becomes pathologically increased, fibrinogen does then leak
into the tissue fluids in sufficient quantities to allow clotting of
these fluids in much the same way that plasma and whole
blood can clot.
Fibrin is formed from fibrinogen under the influence of
thrombin. Fibrinogen – is fibrilar protein, which consists of
fibrin monomer and four fibrinopeptides (2A and 2B).
Fibrinopeptides disturb fibrin monomer to polymerize.
Thrombin rifts fibrinopeptides from fibrinogen and form
fibrin monomer. Polymerization of fibrin monomer starts and
fibrin S is formed, which is soluble. With the help of F. XIII
(fibrin stabilizing factor) and calcium in S molecule covalent
connections are formed and fibrin S turns to insoluble fibrin I.

129
 Fibrinogen (А2В2)

Thrombin (F.ІІ)

Fibrin monomer
+2А+2В

Fibrin polymer soluble

(fibrin S)

F. ХІІІ F. ХІІІа
Fibrin insoluble
(fibrin I)

So, the result of the third stage is frormation of fibrin.

The clot is composed of a meshwork of fibrin fibers running in
all directions and entrapping blood cells, platelets, and
plasma.The fibrin fibers also adhere to damaged surfaces of
blood vessels; therefore, the blood clot becomes adherent to
any vascular opening and thereby prevents further blood loss.
Formed blood elements mesh in fibrin fibers. But this
ball of fibers is soft. That’s why the next step of the process is
consolidation of thrombus. Packing of thrombus occurs due to
contraction of protein thrombocystein (PF-6). After retraction
clot is packed in 2 times, serum is removed from it and it
becomes compact and plasma can not pass through it.
Platelets are necessary for clot retraction to occur.
Therefore, failure of clot retraction is an indication that the
number of platelets in the circulating blood might be low.
Electron micrographs of platelets in blood clots show that they
become attached to the fibrin fibers in such a way that they

130
actually bond different fibers together. Furthermore, platelets
entrapped in the clot continue to release procoagulant
substances, one of the most important of which is fibrin-
stabilizing factor, which causes more and more cross-linking
bonds between adjacent fibrin fibers. In addition, the platelets
themselves contribute directly to clot contraction by activating
platelet thrombosthenin, actin, and myosin molecules, which
are all contractile proteins in the platelets and cause strong
contraction of the platelet spicules attached to the fibrin. This
also helps compress the fibrin meshwork into a smaller mass.
The contraction is activated and accelerated by thrombin as
well as by calcium ions released from calcium stores in the
mitochondria, endoplasmic reticulum, and Golgi apparatus of
the platelets. As the clot retracts, the edges of the broken blood
vessel are pulled together, thus contributing still further to the
ultimate state of hemostasis.
Retraction last for 2-3 hours. After some time clot starts
to spring with fibroblasts. This occurs under the influence of
thrombocytes growth factor. Integrity of the damage spot of
vessel is restored.

Figure 5.2 – Red thrombus: erythrocytes and fibrin fiber

131
 Role of Calcium Ions in the Intrinsic
and Extrinsic Pathways

Except for the first two steps in the intrinsic pathway,

calcium ions are required for promotion or acceleration of all
the blood-clotting reactions. Therefore, in the absence of
calcium ions, blood clotting by either pathway does not occur.
In the living body, the calcium ion concentration seldom falls
low enough to significantly affect the kinetics of blood clotting.
But, when blood is removed from a person, it can be prevented
from clotting by reducing the calcium ion concentration below
the threshold level for clotting, either by deionizing the calcium
by causing it to react with substances such as citrate ion or by
precipitating the calcium with substances such as oxalate ion.

Fibrinolysis

Fibrinolysis – disintegration of fibrin. Disintegration of

fibrin starts at the same time with thrombus retraction.
Disintegration is performed by fibrinolysis system.

Content of fibrinolytic system

1 Plasminogen (profibrinolysin) – not-active proteolytic
enzyme, which is always in blood plasma.
2 Plasmin (fibrinolysin) – active enzyme, which is produced
in the result of effect of active proteases on plasminogen.
3 Activators of fibrinolysis – substances that are proteases or
provide proteases synthesis.
4 Inhibitors of fibrinolysis.

There are internal and external mechanisms of

fibrinolysis activation.

132
 Internal mechanism includes activation of F. XII and
formation of calicrein, which cause large amount of
fibrinolysis activators to appear in blood.
Internal mechanism is associated with transport of ready
fibrinolysis activators into the blood.

Activation of Plasminogen to Form Plasmin: Then

Lysis of Clots
When a clot is formed, a large amount of plasminogen is
trapped in the clot along with other plasma proteins. This will
not become plasmin or cause lysis of the clot until it is
activated. The injured tissues and vascular endothelium very
slowly release a powerful activator called tissue plasminogen
activator (t-PA) that a few days later, after the clot has stopped
the bleeding, eventually converts plasminogen to plasmin,
which in turn removes the remaining unnecessary blood clot. In
fact, many small blood vessels in which blood flow has been
blocked by clots are reopened by this mechanism.

A Plasmin Inhibitor, Alpha2-Antiplasmin Plasmin not only

destroys fibrin threads but also functions as a proteolytic
enzyme to digest fibrinogen and a number of other clotting
factors as well. Small amounts of plasmin are formed in the
blood all the time, which could seriously impede the activation
of the clotting system were it not for the fact that the blood also
contains another factor, alph 2 -antiplasmin, t hut binds with
plasmin and inhibits it. Therefore, the rule of plasmin
formation must rise above a certain critical level before it
becomes effective.

133
Internal External
mechanism mechanism

Plasminogen
factor XIIa,
calicrein

Activators Activators
endothelial
tissue
blood
bacterial
Plasmin (streptokinase)
renal
(urokinase)

Inhibitors

After activation plasmin is blocked by antiplasmin, that’s why

it works only locally in the blood clot.

Anticoagulative system
Blood fluidity is maintained by several mechanisms such as:
1) smooth surface of vessel wall endothelium;
2) negative charge of vessel wall and formed
blood elements, so they repel from each other;
3) thin fibrin layer on vessel wall, which absorb
factors of blood clotting, especially thrombin;
4) synthesis of prostacyclin by endothelium,
which is inhibitor of aggregation;
5) ability of endothelium to synthesize and fix
antithrombin III;

134
6) presence of anticoagulants in the bloodstream.

Classification of anticoagulants
1 Primary (always present in plasma):
antithrombin III, heparin, α1-antithropsin, α2-macroglobulin.
2 Secondary (they are formed in the process of
clotting): antithrombin I, products of fibrinolysis.
Antithrombin III is α2-globulin of blood plasma. Its
concentration in blood plasma is 240 mg/ml. It is 75% out of
all anticoagulative reserves of blood. It inactivates thrombin (F.
IIa), XIIa, XIa, Xa, IXa.
Heparin – sulphuretted polysugar. Active only with
antithrombin III. It provides fixation of antithrombin III on the
surface of endothelium that increases its activity in hundred
times.
Significance of the Plasmin System The lysis of blood
clots allows slow clearing (over a period of several days) of
extraneous clotted blood in the tissues and sometimes allows
reopening of clotted vessels. An especially important function
of the plasmin system is to remove very minute clots from the
millions of tiny peripheral vessels that eventually would all
become occluded were there no way to cleanse them.

135
 Questions for self-control
1 Functions of haemostasis system.
2 Mechanisms of haemostasis.
3 Role of vessel wall in haemostasis.
4 Functions of thrombocytes.
5 Stages of vessel-thrombocytory haemostasis.
6 Spasm of vessels. Its types.
7 Mechanisms and stages of adhesion.
8 Definition of thrombus aggregation. Stages and mechanisms
of aggregation.
9 Consolidation of thrombus.
10 Factors of blood clotting.
11 Stages of blood clotting.
12 Formation of tissue and blood prothrombinase. Role of
prothrombinase.
13 Role of calicrein-kinine system in haemostasis.
14 Formation of thrombin.
15 Formation of fibrin.
16 Fibrinolysis.
17 Anticoagulative system.

Tests for self-control

1 Which substances out of the following are factors of blood
clotting:
a) antithrombin III;
b) fibrinstabilizing factor;
c) bradykinine;
d) serotonin;
e) angiotensin III;
f) calidine;

136
 g) magnesium ions;
h) no correct answer?
2 Which substances comprise anticoagulative system:
a) proconvertin;
b) prothrombin;
c) Hageman’s factor;
d) serotonin;
e) calidine;
f) antithrombin III;
g) calcium ions;
h) no correct answer?

3 Which substances are factors of blood clotting:

a) heparin;
b) histamine;
c) profibrinolysin;
d) vitamin K;
e) vitamin E;
f) fibrinogen;
g) magnesium ions;
h) no correct answer?

4 Which substances comprise anticoagulative system:

a) antithrombin III;
b) proaccelerin;
c) fibrinstabilizing factor;
d) histamine;
e) antihaemophilic globulin;
f) bradykinine;
g) calcium ions;
h) no correct answer?

5 Which substances are factors of blood clotting:

a) proconvertin;

137
 b) prothrombin;
c) Hageman’s factor;
d) serotonin;
e) calidine;
f)antithrombin III;
g) calcium ions;
h) no correct answer?

6 Which substances comprise fibrinolytic system:

a) heparin;
b) fibrinogen;
c) histamine;
d) plasmin;
e) proaccelerin;
f) vitamin E;
g) calcium ions;
h) no correct answer?

7 First stage of blood clotting results in the formation of:

a) thrombin;
b) fibrin;
c) calicrein;
d) plasmin;
e) ptothrombinase;
f) no correct answer.

8 Second stage of blood clotting results in the formation of:

a) thrombin;
b) fibrin;
c) calicrein;
d) plasmin;
e) prothrombinase;
f) no correct answer.

138
9 Third stage of blood clotting results in the formation of:
a) thrombin;
b) fibrin;
c) calicrein;
d) plasmin;
e) prothrombinase;
f) no correct answer.

10 Formation of thrombin is the result of:

a) first stage of blood clotting;
b) second stage of blood clotting;
c) third stage of blood clotting;
d) activation of anticoagulative system;
e) activation of fibrinolytic system;
f) no correct answer.

11 Secretion of heparin is the result of:

a) first stage of blood clotting;
b) second stage of blood clotting;
c) third stage of blood clotting;
d) activation of anticoagulative system;
e) activation of fibrinolytic system;
f) no correct answer.

12 Which enzyme induces formation of thrombin:

a) Hageman’s factor;
b) tissue thromboplastin;
c) fibrinolysin;
d) proconvertin;
e) calicrein;
f) plasmin;
g) prothrombinase;

139
 h) no correct answer?

13 Which processes participate stoppage of bleeding in vessels

with diameter less than 100 mkm:
a) spasm of arteries;
b) adhesion of thrombocytes;
c) aggregation of thrombocytes;
d) formation of fibrin;
e) no correct answer?

14 Thrombin induces transformation of:

a) proconvertin into convertin;
b) proaccelerin into accelerin;
c) factor V into factor VII;
d) factor VII into factor VIII;
e) factor VIII into factor IX;
f) factor IX into factor X;
g) fibrinogen into fibrin.

15 Which are the two mechanisms of bleeding stoppage

(haemostasis):
a) vessel-thrombocytory haemostasis;
b) vessel-leukocytory haemostasis;
c) vessel-erythrocytory haemostasis;
d) leukocytory haemostasis;
e) thrombocytory haemostasis;
f) erythrocytory haemostasis;
g) coagulative haemostasis?

140
 PRACTICAL WORKS

Practical work #1. Definition of hematocrit index

Hematocrit index (hematocrit) characterizes the

percentage volume of formed elements (erythrocytes) in blood.
There are venous, capillary and arterial hematocrits, because
the volume of formed blood elements, especially erythrocytes,
is different in different parts of the bloodstream. The lowest
hematocrit is in the arterial blood.
Objectives: to define the percentage volume of formed
elements (erythrocytes) in capillary blood and estimate its
value.
Requirements for work: hematocrit capillary or micropipette,
centrifuge with 8000 rotations per minute, plasticine, aqueous
solution of heparin (with activity 5000 OD/ml), 96% alcohol,
2% alcohol solution of iodine, cotton wool.

 to wash calibrated hematocrit pipette with heparin

solution;
 to blow pipette;
 to dry pipette and fill with blood on 7/8 long;
 to fix the opening of pipette with plasticine and to
centrifuge for 5 minutes with 8000 rotations per minute;
 to define the percentage volume of formed elements
comparatively to the general blood volume, that is
hematocrit value.
Recommendations for writing down the results
Write down hematocrit value in percents.
Answer following questions in conclusion:
 Is the percentage volume of formed element
(erythrocytes) in capillary blood normal?
 What changes in blood system testify increase or
decrease of hematocrit value?

141
Practical work #2. Definition of osmotic resistance of
erythrocytes

Resistance of erythrocytes is the ability of erythrocyte

membrane to resist the destructive action of low osmotic
pressure in solution, where erythrocytes are. In result of such
action haemolysis can occur (degradation of the erythrocytes
membrane).
Resistance of erythrocyte membrane should be studied
relatively to hypotonic solutions of sodium chloride. When
concentration is 0,54% - 0,45% normally only erythrocytes
with the lowest resistance pass through haemolysis (minimal
erythrocytes resistance). In case of further decrease of
concentration of sodium chloride more persistent erythrocytes
pass through haemolysis. When concentration is 0,40% -
0,34% of sodium chloride even most persistent erythrocytes
break down (maximal resistance). Solution becomes
transparent, like varnish (as called laky solution). Breach
between upper and lower limit of resistance is called amplitude
of resistance.
Objectives: to define the borders of changes in osmotic
pressure of blood plasma, when the integrity of erythrocyte
membrane is kept and estimates the durability of the
erythrocyte membrane.
Requirements for work: 7 Vidal’s test-tubes (or plain
laboratory tubes), holder, 0,5% solution of sodium chloride,
distillated water, blood, conserved with 5% solution of sodium
citrate, 3 pipettes with the equal size of the drop openings.

 Make following dilutions with 0,5% solution of sodium

chloride:

142
№ of Number of Number of Concentration
test-tybe 0,5% NaCl distillated of solution
drops water drops
1 25 - 0.5%
2 24 1 0.48%
3 22 3 0.44%
4 20 5 0.40%
5 18 7 0.36%
6 16 9 0.32%
7 14 11 0.28%

 Add 1 drop of conserved blood in every test-tube.

 Accurately mix the content of every tube until it will
get steady color and leave it for 1 hour in a holder.
 In 1 hour time observe the destruction of erythrocyte
membrane (haemolysis). State of blood is estimated by
the grade of color of sodium chloride solution, which is
up to sedimentated erythrocytes in test-tubes.
Recommendations for writing down the results:
 Write down the value of minimal and maximal
erythrocyte resistance.
 Calculate its amplitude.
Answer the following questions in conclusion:
 Do the borders of changes in osmotic pressure, when
erythrocyte membrane keeps its integrity correspond to
normal?
 Is the integrity of erythrocyte membrane normal?
 What testifies increase (or decrease) of the resistance
borders?

143
Practical work #3. Definition of ESR

Blood is a colloid solution and suspension at the same

time. Particles of substances that are suspended in liquid
medium undergo action of inverse forces: gravity force, which
provides sedimentation of particles and diffusion, with the help
of which particles of colloids move.
Rate of particle sedimentation is straightly proportional
to the square radius and difference between the densities of
suspended substance and dissolvent. The charges of particles,
which are in the solution, also play a great role.
Formed elements, which are suspended in the solution
of colloids and bound with them by charges, will sedimentate
in stabilized blood because of their agglomeration. Blood will
divide into two layers: upper is plasma, lower is blood
elements.
Correlation of cholesterol and lecithin in plasma,
content of bilious pigments and bilious acids, changes in
stickiness, pH, characteristics of erythrocytes, and amount of
hemoglobin and so on affect ESR.
Main factors that affect ESR are quantitative and
qualitative changes of proteins in plasma. So, increase of
globulin amount in plasma leads to increase of ESR and
decrease of its amount and increase of albumins amount leads
to decrease of ESR.
ESR gives information about the correlation of plasma
proteins and their electrostatic interaction with erythrocytes.
Normally ESR of men is 2-10 mm/hour, of women – 2-
15 mm/hour.
Objectives: to define erythrocyte sedimentation rate;
estimate correlation between albumins and globulins in blood
plasma.
Requirements for work: Panchenkov’s apparatus, which
includes holder with rubber base, capillaries for the definition

144
of ESR, glass, 5% solution of sodium citrate, 96% ethanol, 2%
alcohol solution of iodine, cotton wool.

Figure 6.1 – Panchenkov’s apparatus

 Wash the capillary with 5% solution of sodium citrate;

 Fill the capillary with mark “R” (reagent) with 5%
solution of sodium citrate and put on the glass;
 Fill the capillary with mark “B” (blood) with blood 2
times and put it on the glass;
 Mix the blood with solution (correlation 4:1), by filling
the capillary with it and putting it back several times;
 Fill the capillary with this mixture till the mark “B”;
 Clean the opening of the capillary from the blood with
cotton wool;
 Put the capillary into the Panchenkov’s apparatus;
 Measure the lighten layer of plasma after 1 hour, which
will be the measure of ESR.
Recommendations for writing down the results:
 Draw down the Panchenkov’s apparatus and capillary
for the measurement of ESR.
 Write down the size of the layer of plasma up to the
erythrocytes, which are sedimentated (mm/hour).

145
 Answer following questions in conclusion:
 Is the ESR normal in the examined blood?
 Is the correlation between albumins and globulins
normal in the blood plasma?
 What changes in blood plasma testify the increase of
ESR?
Practical work #4. Calculation of the erythrocytes
Erythrocytes are counted with the help of Goryaev’s
calculating camera under the microscope. This method is
complicated but regular enough (permissible variation is not up
to 2,5%).
The net rate of calculating camera consists of 225 large
quadrates, 25 out of them are divided into 16 small ones.
The side of small quadrate is 1/20 mm, square is 1/400 mm 2,
the height of camera (the distance between the bottom and the
covering glass) is 1/10 mm. So, the size of the camera upon
the small quadrate is 1/4000 mm3 (1/400•1/10).
Blood for the count of the erythrocytes is diluted in
special mixing tube (melanger) – capillary pipettes with the
ampule dilation. There are marks 0,5 and 101 on the mixing
tubes. Mark 0,5 shows what part of the mixing tube takes this
column of capillary, filled with blood. This volume takes 1/200
of the all volume of the mixing tube. So, blood is dissolved in
200 times. Blood can be diluted in 200 times by other methods.
For example, put 4 ml of 5% solution of sodium citrate in the
test-tube and add 20 ml of blood with micropipette. It is
necessarily to washout the micropipette three times in this
solution, so all blood will get into the tube.
Normally the amount of erythrocytes in men is 4-5•1012,
in women 3,9-4,7•1012.

146
Figure 6.2 – А, B – Goryaev’s calculating camera;
C – camera calculating net :
а – small quadrate;
b – large quadrate

147
 Objectives: to count
erythrocytes; to estimate the
quantity of erythrocytes in the
peripheral blood.
Requirements for work:
microscope, Goryaev’s camera,
covering glass, mixing tube for
the erythrocytes, 3% solution of
sodium chloride, 96% alcohol,
2% alcohol solution of iodine,
cotton wool.

Figure 6.3 – Erythrocyte mixing tube

 To prepare Goryaev’s camera for work:

 perform defatting with alcohol and dry the camera and
the covering glass;
 lap the covering glass to the camera till the appearance
of the Newton’s rings;
 find the net under the large enlargement.
1. Prepare blood for work:
 fill the capillary till the mark of 0,5; clean the opening
from blood with the cotton wool;
 don’t release the blood from the capillary and fill it with
3% solution of sodium chloride till the mark of 101;
 mix the solution with blood in the ampule of mixing
tube (there is a tiny red ball to ease the mixing process).
Blood will be diluted in 200 times.
2. To fill camera with blood:
 blow first two drops of solution out of the capillary on
the cotton wool;

148
  next drops from the ampule solution put in the camera.
Put the tip of the melager on the edge of camera near
the covering glass and blow it out accurately. Solution
will go under the covering glass into the camera and
will fill it. Wait for 1-2 minutes for erythrocytes to
sedimentate on the bottom of the camera.
3. Calculate the amount of the erythrocytes:
 count the amount of erythrocytes in 5 large quadrates of
the net diagonally. Remember Burker’s rule when
counting erythrocytes: in small quadrates count cells,
which are inside the quadrate and on its superior and
left sides. This will predict counting erythrocytes twice.
 calculate the amount of erythrocytes in 1 mkl of blood
by formula:
E   a  4000  200 / 1  5 16
Where E – is the quantity of erythrocytes in 1 mkl;
a – the amount of erythrocytes in 5 large quadrates of net;
5 – the amount of large quadrates;
16 – the amount of small quadrates;
200 – the degree of blood dilution;
1/4000 mm3 – the volume of 1 small quadrate
There is a simplified formula:
E = a•104
- to find the amount of erythrocytes in 1 l of blood by formula
E•106
Recommendations for writing down the results:
Draw down the mixing tube for the erythrocytes.
Write down the process of the erythrocyte counting.
Define the amount of erythrocytes in 1 l of blood.
Answer following questions in conclusion:
Is the amount of erythrocytes normal in the examined blood?

149
Practical work #5. Method of definition of hemoglobin level
in blood (Sali’s method)

Sali method is used for the definition of the hemoglobin

amount in blood.
Principle of this method is the following. After adding
hydrochloric acid hemoglobin transforms into hydrochloric
hematin that has brown color, intensity of which is straightly
proportional to the hemoglobin content. The solution of
hydrochloric hematin is diluted with water till it gets standard
color.
This method of the definition of hemoglobin amount
gives results with accuracy to 10%. Mistake is associated with
the technical problems during definition and possible
subjective mistakes in the definition of color. But this method
is widely spread because it is easy and simple.
Hemometer Sali is used for this definition. It is a holder
with 3 test-tubes. There is 1% solution of hydrochloric
hematin. Middle tube has marks from 0 to 23•10 g/l and it is
for the definition of hemoglobin in the examined blood.
Normally, the amount of hemoglobin in men is 140-160 g/l, in
women – 120-140 g/l.

150
Figure 6.4 – Hemometer Sali; a – tubes with standard solution of muriatic
hematin; b – tube for the definition of hemoglobin
Objectives: to define and estimate the amount of hemoglobin in
the examined blood.
Requirements for work: hemometer Sali, pipette, glass stick,
0,1N solution of hydrochloric acid, distillated water, 96%
ethanol, cotton wool.

 With the help of pipette put 0,1N solution of

hydrochloric acid into the middle tube of hemometer till
the lowest mark (0,2 ml);
 For the definition of hemoglobin put blood into the
capillary till the mark (0,02 ml);
 Clean the opening of the capillary with cotton wool;
 Put the capillary into the tube with acid and carefully
eject the blood from the capillary to the bottom of the
tube;
 Without taking out the capillary from the tube, clean it
with acid from the upper layers;
 Mix blood with acid by shaking the tube;
 Put the tube into the hemometer;
 Leave hemometer for 4-5 minutes. By this time acid
will ruin erythrocyte membrane, transform hemoglobin
into hydrochloric hematin that has brown color;
 Put distillated water into the middle tube, until the color
of the solution in the middle tube will become the same
color with the one in standard tubes;
 Fix the level of solution in the middle tube by the lower
meniscus;
 To get the value in g/l, the value in g% should be
multiplied by 10.
Recommendations for writing down the results:
Draw down hemometer of Sali.

151
Calculate the amount of hemoglobin in the examined blood.
Write down the results in the absolute units.
For example:
Hemometer has graduations in absolute units of hemoglobin.
Result – 15 g%
Calculating: 15 g% • 10 = 150 g/l.
Answer following questions in conclusion:
Is the amount of hemoglobin normal in the examined blood and
what does it testify?

Practical work #6. Calculation of the color index

In clinics 5 indexes of the blood is calculated: color

index, average hemoglobin content in the erythrocyte, average
concentration of hemoglobin in the erythrocyte, average
erythrocyte volume, average diameter of erythrocyte. It is
necessary to define color index in clinical analysis.
The value of this index shows relative hemoglobin
content in every erythrocyte. Normally color index is 0,85 –
1,15. Increase or decrease of it testifies interruption of the
erythrocytes saturation with hemoglobin.

152
 a b c
Figure 6.5 - Nomogram is used for the calculation of color index: a –
normal color index value;
b – hemoglobin content by Sali method (%);
c – number of erythrocytes (in 1 l of blood)

To calculate hemoglobin amount in % the following

operations are performed. For example there is 14 g% of
hemoglobin in blood:
16,7 g% - 100%
14 g% - x x = 14•100 / 16,7 = 84%

Objectives: to define and estimate relative hemoglobin content

in the erythrocyte.
Find the amount of erythrocytes in 1 mkl of blood and amount
of hemoglobin, calculate color index (CI).
If the amount of hemoglobin is in g/l, then CI is calculated
using a formula:
CI = (number of Hb (g/l)•3): (first 3 digits of the erythrocytes
amount)

153
For example, if hemoglobin amount is 140 g/l, erythrocytes –
4,2 • 1012 (4 200 000 000 000), then CI = (140 • 3) : 420 = 1
Recommendations for writing down the results:
Calculate the color index using data from previous practical
works.
Answer following questions in conclusion:
What is the degree of erythrocyte saturation with hemoglobin
and what does it testify?

Practical work #7. Calculation of leukocytes

Leukocytes or white bodies have many variations and perform

different protective high-specified functions. That’s why the
amount of leukocytes varies during a short period of time in the
healthy man (from 4•109/l to 9•109/l).
Dilution of the blood for the calculation of leukocytes is
better to be performed with the help of melanger.

In clinics other
methods of blood
dilutions are also used.
For example, 0,4 ml of
3% solution of acetic
acid, which is painted
with methylene-blue is
put in the dry tube,
then 0,02 ml of blood
is added. Blood gets
by any pipette with
graduation. It is
important to keep
correlation 1:20.

154
Figure 6.6- Melanger for leukocytes

Acetic acid is needed in calculation of leukocytes to cause

hae molysis of erythrocytes, and methylene-blue – for the
contrast of leukocytes nuclei, so for the convenience of
calculation.
Objectives: to calculate the amount of leukocytes in
Goryaev’s camera.
Requirements for work: microscope, lighter, Goryaev’s
camera, covering glass, melanger for leukocytes, holder
with tube, 3% solution of acetic acid, 96% ethanol, cotton
wool.

1. To prepare Goryaev’s camera for work:

 perform defatting with alcohol and dry the camera and
the covering glass;
 lap the covering glass to the camera;
 find the net under the large enlargement.
2. Prepare blood for work:
 fill the capillary till the mark of 0,5; clean the opening
from blood with the cotton wool;
 don’t release the blood from the capillary and fill it with
3% solution of acetic acid till the mark of 11;
 mix the solution with blood in the ampule of mixing
tube (there is a tiny red ball to ease the mixing process).
Blood will be diluted in 20 times.
3. To fill camera with blood:
 blow first two drops of solution out of the capillary on
the cotton wool;
 the next drop from the ampule solution is put in the
camera. Put the tip of the melager on the edge of
camera near the covering glass and blow it out
accurately. Solution will go under the covering glass

155
 into the camera and will fill it. Wait for 1-2 minutes for
leukocytes to sedimentate on the bottom of the camera.
4. Count the amount of the leukocytes:
 count the amount of erythrocytes in 100 large
quadrates. For better accuracy calculation should be
performed on the whole area of net, starting from the
left edge.
 calculate the amount of erythrocytes in 1 mkl of blood
by formula:

L = (a • 4000 • 20) / 1 • 100 • 16

Where L – the amount of leukocytes in 1 mkl;

a – the amount of leukocytes in 100 large quadrates of net;
100 – the amount of large quadrates;
16 – the amount of small quadrates;
20 – the degree of blood dilution;
1/4000 mm3 – the volume of 1 small quadrate
There is a simplified formula:
L = (a:2) • 102
- to find the number of leukocytes in 1 l of blood
L • 106
Recommendations for writing down the results:
Draw down the melanger for leukocytes.
Count the amount of leukocytes in 100 quadrates of net.
Calculate the number of leukocytes in 1 ml and in 1 l of
blood.
Answer following questions in conclusion:
Is the amount of leukocytes normal in examined blood and
what does it testify?

Practical work #8. Definition of the blood group by

ABO system with the help of coliclones

156
 For the definition of the blood group in any system the
same principle is used: providing conditions for
erythrocytes agglutination in the medium of standard
isohemagglutinating serums or coliclones, that have high
titre of antibodies to the examined antigens of erythrocytes.
Objectives: To define the blood group by ABO system.
Requirements for work: white plate, pipettes, glass, pencil
for glass, examined blood, closed tubes with solutions of
coliclones anti-A and anti-B, isotonic solution of sodium
chloride.

 divide dry white plate on 4 sectors with glass-pencil.

 make notes “anti-A”, ”anti-B”.
 with the help of pipettes put drops (0,1 ml) of
coliclones anti-A and anti-B in the accordant sector.
Make a string using the same pipette (diameter of the
string should not be less than 1,5 – 2 cm).
 put one drop of the examined blood on the glass.
 using the angles of another glass put blood (0,01 ml) in
the drops of coliclones. Angles of glass should be
different for different coliclones.
 mix blood with the drop of coliclone with the angle of
glass. Correlation between blood and coliclone should
be 1:10 (mixed drop has pink color).
 observe the reaction on the plate during 2,5 minutes.
Results and control
1. Agglutination does not occur with coliclone anti-A and
with coliclone anti-B. So, examined erythrocytes do not
have antigens A and B, and blood belongs to group I (0,
αβ).
2. Agglutination occurred only with coliclone anti-A. So,
examined erythrocytes have only antigen A and blood
belongs to group II (A, β).

157
 3. Agglutination occurred only with colicone anti-B. So,
examined erythrocytes have only antigen B and blood
belongs to group III (B, α).
4. Agglutination of erythrocytes is observed in both drops
of coliclones. So, examined erythrocytes have both
antigens A and B and blood belongs to group IV (AB).
It should be mentioned that all processes that occur after 2,5
minutes after mixing will not be connected with specific
agglutination, which is examined and those can have other
reasons. False agglutination can occur when erythrocytes will
gather in monetary column. This agglutination can be easily
discerned from the real one if added 1-2 drops of isotonic
solution of sodium chloride to 1 drop of blood. False
agglutination will disappear in this case.
Recommendation for writing down the results:
Write down the results of agglutination reaction with coliclones
anti-A and anti-B in the following chart:

coliclone
Anti-A Anti-B
blood
І (0, αβ)
ІІ (А, β)
ІІІ (В, α)
ІV (АВ)

Answer the following questions in conclusion:

What means presence/absence of agglutination of the examined
blood with coliclone anti-A?
What means presence/absence of agglutination of the examined
blood with coliclone anti-B?

158
Practical work #9. Definition of blood group by ABO
system with the help of standard serums

Objectives: to define a blood group by ABO system.

Requirements for work: chart boards for the definition of blood
group, pipettes, glass, examined blood, standard serums,
isotonic solution of sodium chloride.

 Put one drop (0,1 ml) of standard serum of I, II, III

group on the surface of the chart board in the
corresponding sector
 Put one drop of the examined blood on the glass with
the help of a pipette
 Using another glass put part of blood (0,01 ml) in each
drop of blood serum. Angles of glass for every serum
should be different.
 Mix blood with serum using the same angle of glass.
Correlation of blood and serum should be 1:10 (mixed
drop should have pink color).
 Observe the reaction on the chart board during 5
minutes.
Results and control
1. Agglutination is absent in all drops of serums. So, we
can suggest that examined erythrocytes do not have
antigens A and B and blood belongs to group I (0, αβ).
2. Agglutination occurred with serums of I and III group.
So, we can suggest that erythrocytes have antigen A
and blood belongs to group II (A, β).
3. Agglutination occurred with serums of I and II group.
So, we can suggest that erythrocytes have antigen B and
blood belongs to group II (B, α).
4. Agglutination is observed in all serums. So, we can
suggest that erythrocytes have both antigens A and B
and blood belongs to group IV (AB). In this case serum

159
 of group IV is put on the plate and if agglutination does
not occur, blood belongs to group IV.

Recommendation for writing down the results:

Write down a chart with the results of agglutination reaction
with serums of different groups:

serum
І (αβ) ІІ (β) ІІІ (α) ІV(-)
blood
І (0) – – – –
ІІ (А) + – + –
ІІІ (В) + + – –
ІV (АВ) + + + –

Answer following questions in conclusion:

What means presence/absence of agglutination of the examined
blood with serums of different groups?

Practical work #10. Definition of the blood group by CDE

system by method of Zotikov-Donsky

Objectives: to define blood group by CDE blood system.

Requirements for work: white plate, 4 pipettes, glass,
examined blood, standard rhesus-positive – (Rh+), standard
rhesus-negative – (Rh-) erythrocytes, standard antirhesus
serum, and pencil for glass.

 divide the plate with the pencil for glass into two parts.
 put one drop of standard antirhesus serum on the one
part of plate, make it plane (with diameter less than 2
cm).

160
  put the examined blood on the glass. Using another
glass put part of blood in drop of serum and mixes it.
Correlation of blood and serum should be 1:10.
There are two variants of reaction:
1. If agglutination occurred that means that there are
antigens CDE in the examined blood, so blood is
rhesus-positive. To confirm the result, control reaction
should be done.
Put 2 drops of standard serums on the other part of the
plate and with the help of glass mix standard
erythrocytes Rh+ and Rh-. If agglutination will not occur
in the drop with Rh- erythrocytes and will occur in the
drop with Rh+ erythrocytes, that means that serum is
reacting correctly. So, results show that blood includes
antigen CDE.
2. If agglutination does not occur, than after control
reaction we can say that blood does not include
antigens CDE. So it is rhesus-negative.
Recommendations for writing down the results:
Draw and describe observed reaction.
Answer following questions in conclusion:
What does agglutination mean when examined and controlled;
is the examined blood rhesus-positive or rhesus-negative?

Practical work #11. Blood test for blood group

compatibility

This test can elicit high antibodies titre in recipient’s

blood versus antigens, that are in donor’s erythrocytes and they
do not belong to main group systems.
Test is performed before the blood transfusion when blood
group by ABO and CDE systems is known. So this test elicits
donor’s and recipient’s blood compatibility by not a main
blood systems.

161
 Objectives: to make a test and to estimate blood
compatibility.
Requirements for work: Petri dish, mirror, pipettes, glass,
serum (plasma) of recipient, blood (erythrocytes) of donor.

 put 1 drop of recipient’s serum (plasma) in Petri dish.

Diameter of drop should be not smaller than 1,5-2 cm.
 put 1 drop of donor’s blood on the glass and put part of
blood into serum using other glass (correlation 1:20).
 put Petri dish on the mirror and observe for 5 minutes.
Recommendations for writing down the results:
Describe the results of the test.
Answer following questions in conclusion:
What does agglutination mean in the test; is the donor’s and
recipient’s blood compatible by not main blood systems?
Practical work #12. Bleeding prolongation test

Duration of bleeding is the time from the moment of the injury

till the moment of bleeding stops. This index gives an idea
about functional balance of components of blood coagulation
system, and about functional activity and quantity of
thrombocytes.
Objectives: to define and estimate the duration of bleeding.
Requirements for work: clock, strings of blotting paper, needle,
96% ethanol, 2% alcohol solution of iodine, cotton wool.

 rub a finger with 96% ethanol and check the time of the
injury. Prick should be done with the whole length of
the needle.
 clean the blood every 30 seconds with the blotting
paper.
 check the time when no blood will be on the paper.
Recommendations for writing down the results:

162
Write down the time when the blood is stopped. Describe
changes of the blood splash diameter on the paper after every
clean.
Answer following questions in conclusion:
Is the bleeding duration normal? What does it testify?

Practical work #13. Definition of capillary resistance

Vessel-thrombocytory haemostasis is the first stage of blood

clotting. With the help of this process small vessel injuries can
be liquidated in short period of time. Thrombocytes constantly
control capillaries permeability. Method that is described
allows defining if thrombocytes perform their protective
function when blood pressure is increased. The degree of
capillary protection depends on the characteristics of capillary
endothelium and the number of thrombocytes in blood.
Objectives: to estimate capillaries resistance to the increase of
blood pressure.
Requirements for work: Rive-Rochi apparatus, clock.

 draw a circle with diameter 5 cm on the skin of the

internal surface of the forearm.
 put the cuff from the manometer on the arm and make
the pressure in it 12 – 13,3 kPa (90 – 100 mmHg).
 hold the pressure in the cuff for 5 minutes.
 wait till the blood circulation in the hand would be
restored. Count the number of petechiae (small
hemorrhages) on the marked surfaces. Pay attention to
the diameters of the petechiae.
 normally the number of petechiae should not be more
than 10 and diameter of it – 1 mm. In case of weak
positive reaction – 20-30, strong positive – more than
30 petechiae
Recommendations for writing down the results:

163
Write down the number and diameters of petechiae.
Answer following questions in conclusion:
Is the capillary resistance of the patient normal?

Practical work #14. Calculation of thrombocytes quantity

in blood

There are direct and indirect methods of counting

thrombocytes. To conduct the last one, they use definition of
quantity of blood platelets in the tinted smear. This index
correlates with the quantity of erythrocytes and in blood
analysis is written as 1:1000. After this, absolute quantity of
thrombocytes is calculated in 1 l of blood. Direct method is
more accurate. Examination is performed in Goryaev’s camera
immediately after taking the blood. To stabilize the blood, the
following solutions can be used: 5-7% trilone B, 1%
ammonium oxalate with methylene-blue and others.
Calculation methods are also different: using phase-contrast
and luminescent microscopy.
Objectives: to calculate and estimate the quantity of
thrombocytes in the examined blood.
Requirements for work: microscope, lighter, mixer for the red
blood, Goryaev’s camera, covering glass, holder with test-tube,
Petri dish with wet filtrating paper (wet camera). 1% solution
of ammonium oxalate, needle, 96% ethanol, 2% alcohol
solution of iodine, cotton wool.

Principle for calculation of thrombocytes is similar to the one

which is used for the definition of erythrocytes amount. But
thrombocytes are degrading fast if we were to retire them from
the bloodstream, that’s why the blood should be taken as fast as
possible and the mixer should be wet. So, mixer should be
partially filled with stabilizing liquid from the test-tube. Prick
the finger with the needle and put the ending of the mixer into

164
the blood, fill it till the mark 0,5. Check for abcence of air in
the end of the mixer, so stabilizing liquid will not drop on the
blood on finger. So, mixer should be held horizontally. Fill the
mixer with blood, put the ending of the mixer into the test-tube
with liquid and fill it till the mark 101. Mix the content of the
melanger and leave it in the horizontal position for 10 minutes
for haemolysis of erythrocytes to occur. Prepare the calculating
camera. Fill it with solution from melanger and put in Petri
dish for 5 minutes. It is necessary for the thrombocytes to settle
on the bottom. Than take it out of the wet camera, pur under
the microscope and calculate thrombocytes in 25 large
quadrates of the net (25 • 16 = 400 small quadrates).
Recommendation for writing down the results:
Number of thrombocytes in 1 mkl is calculated by the
following formula:
X = (a • 400 • 200) / 400
Where x – the quantity of thrombocytes in 1 mkl;
a – the quantity of thrombocytes in 400 small
quadrates;
200 – degree of blood dilution;
400 – multiplier, which turns the result to the volume of
1 mkl (from the volume of small quadrate).
Practically the amount of thrombocytes in 400 small quadrates
is multiplied by 2000 and then again by 10 6 and we have the
amount of thrombocytes in 1 l.
Answer following questions in conclusion:
Is the quantity of thrombocytes normal in the examined blood?

Practical work #15. Definition of the total time of blood

clotting

Objectives: to define the total time of blood clotting and

estimate it.

165
Requirements for work: glass, glass hook, clock, needle, 96%
ethanol, 2% alcohol solution of iodine, cotton wool.
 take blood from a rat’s tail, put it on the glass and check
the time.
 take out the content of drop with the interval 20-30 sec
(hook should be held vertically), wait, till fibrin fiber
will drag after the hook.
 check the time again and consider it as the moment the
clotting started.
 put the hook into the blood, drag the drop on the glass
horizontally with the same interval. Check the time as
soon as clot will be dragged after the hook, which
corresponds to the end of clotting.
Recommendations for the writing down the results:
Write down the time the clotting started and the time the
clotting ended.
Answer following questions in conclusion:
Is the time of blood clotting normal? What does it testify?

166
Appendix I

I. Blood plasma content

Inorganic part:
Fe (iron) 8,53 - 28,06 mkmol/l
K (potassium) 3,8 - 5,2 mmol/l
Na (sodium) 138-217 mkmol/l
Ca (calcium) 0,75 - 2,5 mkmol/l
Mg (magnesium) 0,78 – 0,91 mkmol/l
P (phosphorus) 0,646 - 1,292 mkmol/l
Chlorides of blood 97 - 108 mkmol/l
Filtrate nitrogen (not-protein) 14,28 - 25 mkmol/l
Urea 3,33 - 8,32 mmol/l
Creatinine 53 - 106,1 mkmol/l
Creatine Men 15,25 - 45,75 mkmol/l
Women 45,75 - 76,25 mkmol/l
Uric acid Men 0,12 - 0,38 mkmol/l
Women 0,12 - 0,46 mkmol/l
Organic part:
Total protein 65 – 85 g/l
Albumins 35 – 50 g/l
(52 – 65%)
Lactatedehydrogenase (LDH) < 7 mmol (hour/l)
Aldolase 0,2 – 1,2 mmol (hour/l)
α-amilase (diastase of blood) 12 – 32 g/l (hour/l)
Aspartateaminotransferase (AST) 0,1 – 0,45 mmol (hour/l)
Alaninaminotransferase (ALT) 0,1 – 0,68 mmol (hour/l)
Cholinesterase 160 – 340 mol (hour/l)
Basic phosphatase 0,5 – 1,3 mmol (hour/l)
Creatinkinase 0,152–0,305mmol (hour/l)
Creatinphosphokinase (KPK) to 1,2 mmol
Lipase 0,4 – 30 mmol (hour/l)
Globulins 3 – 35 g/l (35 – 48%)

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Total bilirubin 8,5 – 20,5 mkmol/l
free bilirubin
(indirect, not conjugated) 1,7 – 17,11 mkmol/l
conjugated bilirubin (direct) 0,86 – 5,1 mkmol/l
Lipids (total amount) 5 – 7 g/l
Triglicerids 0,59 – 1,77 mmol/l
Total cholesterol 2,97 – 8,79 mmol/l
Lipoproteins of very low density 1,5 – 2,0 g/l
(0,63 -0,69 mmol/l)
low density 4,5 g/l
(3,06 – 3,14 mmol/l)
high density 1,25 – 6,5 g/l
(1,13 – 1,15 mmol/l)
Chylomicrons 0 – 0,5 g/l
(0 – 0,1 mmol/l)
Glucose of the blood 3,3 – 5,5 mmol/l
Glycolized hemoglobin 4 – 7%

II. Normal Values for Erythrocyte and Leukocyte

Measurements

Hemoglobin 13–18 g/dL (males);

12–16 g/dL (females)
Hematocrit 42–52% (males);
37–48% (females)
Erythrocyte count (male) 4.5–6.0 × 106/mm3
(females) 4.0–5.5 ×106/mm3
Leukocyte count 5 × 103–10 × 103/mm3
Differential Leukocyte Count
Neutrophils 55–75%
Eosinophils 2–4%
Basophils 0.5–1%
Lymphocytes 20–40%
Monocytes 3–8%

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Appendix II

Answers on tests for self-control:

Chapter 1
1 – e, 2 – a, d, g, 3 – d, 4 – b, 5 – b, c, 6 – b, d 7 – f, 8 – b, d,
9 – c, 10 – a, d, f, g, 11 – a, 12 – b, 13 – c, 14 – c, 15 – c, d.

Capter 2
1 – e, 2 – a, 3 – c, 4 – b, d, 5 – c, 6 – c, 7 – b, c, 8 – c, 9 – a, c,
d, e, 10 – a, 11 – c, 12 – e, 13 – a, 14 – c, 15 – d.

Chapter 3
1 – a, c, 2 – c, d, 3 – a, c, 4 – a, 5 – c, d, 6 – b, 7 – a, 8 – c, d,
9 – b, c, 10 – b, d, 11 – a, b, c, e, 12 – b, 13 – c, 14 – d, 15 – c.

Chapter 4
1 – a, b, d, e, g, 2 – b, 3 – d, e, g, 4 – a, f, 5 – d, 6 – c, 7 – a,
8 – d, 9 – e, 10 – c.

Chapter 5
1 – b, d, 2 – f, 3 – f, 4 – a, 5 – a, b, c, g, 6 – d, 7 – e, 8 – a,
9 – b, 10 – b, 11 – d, 12 – g, 13 – a, b, c, 14 – g, 15 – a, g.

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