HIGHER STATE EDUCATIONAL INSTITUTION OF UKRAINE

“UKRAINIAN MEDICAL STOMATOLOGICAL ACADEMY”

PHYSIOLOGY DEPARTMENT

ZAPOROZHETS T.N., TKACHENKO E.V.

PHYSIOLOGY

METHODICAL INSTRUCTIONS TO PRACTICAL CLASSES FOR STUDENTS

OF MEDICAL AND DENTAL PROPHILES

PART 2. VISCERAL SYSTEMS: “BLOOD PHYSIOLOGY”

МІНІСТЕРСТВО ОХОРОНИ ЗДОРОВ'Я УКРАЇНИ

ВИЩИЙ ДЕРЖАВНИЙ НАВЧАЛЬНИЙ ЗАКЛАД УКРАЇНИ
“УКРАЇНСЬКА МЕДИЧНА СТОМАТОЛОГІЧНА АКАДЕМІЯ”

КАФЕДРА ФІЗІОЛОГІЇ

ЗАПОРОЖЕЦЬ Т.М., ТКАЧЕНКО О.В.

ФІЗІОЛОГІЯ

НАВЧАЛЬНИЙ ПОСІБНИК ДЛЯ СТУДЕНТІВ МЕДИЧНОГО ТА

СТОМАТОЛОГІЧНОГО ФАКУЛЬТЕТІВ

ЧАСТИНА 2. ФІЗІОЛОГІЯ ВІСЦЕРАЛЬНИХ СИСТЕМ:

«ФІЗІОЛОГІЯ КРОВІ»

ПОЛТАВА 2009
 Approved by
the Central methodical commission
the protocol N.4 from 19.02.2009

It has been composed by Zaporozhets T.N., d.med.sci., prof., Tkachenko E.V.,

cand.med.sci., assistant
Reviewers:
The doctor of medical sciences, professor Kostenko V.A., the head of pathological
physiology department of higher state educational institution of Ukraine “Ukrainian
Medical Stomatological Academy”, Poltava.
Cand.Phil.Sci Znamenskaya I.V., the higher lecturer of foreign languages
department of higher state educational institution of Ukraine “Ukrainian Medical
Stomatological Academy”, Poltava.
The manual contains theoretical material and material for practical classes, questions
for students' self-preparing as well as tests and tasks (with answers) for training to
Krok-1. It is illustrated by figures and tables.

Затверджено Центральною
методичною комісією
Протокол № 4 від 19.02.2009 р.

Склали: Запорожець Т.М., д.мед.н., професор. Ткаченко О.В., к.мед.н.,

асистент
Рецензенти:
Доктор медичних наук, професор Костенко В.О., завідувач кафедри
патологічної фізіології Вищого державного навчального закладу України
“Українська медична стоматологічна академія”, м.Полтава
Кандидат філологічних наук Знаменська І.В., старший викладач кафедри
іноземних мов Вищого державного навчального закладу України “Українська
медична стоматологічна академія”, м.Полтава

Посібник містить теоретичний матеріал та практичні завдання, питання до

самопідготовки студентів, а також навчальні тести та задачі (із відповідями) для
підготовки до ліцензійного іспиту з “Крок-1”. Посібник вдало ілюстрований
тематичними малюнками і таблицями.

2
 CONTENT

СONTENT MODULE 11: BLOOD SYSTEM PHYSIOLOGY ............................. 5

LESSON 31 .............................................................................................................. 5
BLOOD PHYSICAL-CHEMICAL FEATURES INVESTIGATION ..................... 5
LESSON 32 ............................................................................................................ 18
ERYTHROCYTES NUMBER AND HEMOGLOBIN CONCENTRATION
INVESTIGATION ................................................................................................. 18
LESSON 33 ............................................................................................................ 39
BLOOD GROUPS BELONGING INVESTIGATION ......................................... 39
LESSON 34 ............................................................................................................ 48
LEUCOCYTES NUMBER, LEUCOCYTIC FORMULE INVESTIGATION ..... 48
LESSON 35 ............................................................................................................ 80
PLATELETS AND VASCULAR-PLATELET HEMOSTASIS INVESTIGATION
................................................................................................................................ 80
LESSON 36 ............................................................................................................ 91
BLOOD COAGULATION INVESTIGATION..................................................... 91
LESSON 37 .......................................................................................................... 101
DIFFERENTIATED COAGULOGRAM. DISSEMINATED INTRAVASCULAR
COAGULATION (DIC) SYNDROME ............................................................... 101
LESSON 38 .......................................................................................................... 112
FIBRINOLYSIS AND ANTICOAGULANTS. BLOOD COAGULATION AND
FIBRINOLYSIS REGULATION ........................................................................ 112
LESSON 39 .......................................................................................................... 122
TOTAL BLOOD .................................................................................................. 122
LESSON 40 .......................................................................................................... 133
PRACTICAL SKILLS ON BLOOD SYSTEM PHYSIOLOGY......................... 133
GLOSSARY ......................................................................................................... 133
BLOOD SYSTEM PHYSIOLOGY ..................................................................... 133

3
TESTS ON BLOOD PHYSIOLOGY .................................................................. 139

4
 СONTENT MODULE 11: BLOOD SYSTEM PHYSIOLOGY

“Blood reflects like in mirror

all processes taking place
in alive organism”.

LESSON 31
BLOOD PHYSICAL-CHEMICAL FEATURES INVESTIGATION

1. The topic studied actuality. Blood is characterized by lots of constants that can
be either very stable or hard (so, their change even a bit can lead to serious, irreversible
changes or even to death – pH, plasma ionic content, osmotic and oncotic pressures,
plasma proteinic content, oxygen partial pressure, glucose content et al.) or unstable
(they can vary in wide ranges and these variations do not lead to any serious results in
organism functioning – circulating blood volume, formed elements content,
hemoglobin level, blood viscosity, velocity sedimentation rate et al.). Blood system
belongs to one of the most sensitive indicators testifying about organism state both
under norm and pathology. Oral cavity also can suffer at blood pathological conditions.

2. Study aims:
To know: blood content and its functions in organism, blood constants and their
significance in clinics, in part, in stomatological one; blood main components,
hematocrit, blood buffer systems, acidosis, alkalosis, erythrocytes osmotic resistance,
velocity sedimentation rate and affecting factors.
To be able to: estimate erythrocytes osmotic resistance, velocity sedimentation rate
and assess the results received.

3. Pre-auditory self-work materials.

3.1.Basic knowledge, skills, experiences, necessary for study the topic:

Subject To know To be able to
Bioinorganic, Data about buffer systems,
bioorganic, blood proteins, osmotically-
physical-colloid active substances
chemistry
Medical Data about buffer systems, Assess given indexes of acid-
biological blood proteins alkaline equilibrium and make a
chemistry conclusion about possible reasons
and developmental mechanisms
Biophysics Data about oncotic and Count hematocrit, velocity
osmotic pressure, hematocrit, sedimentation rate and blood
velocity sedimentation rate, viscosity, erythrocytes osmotic
blood viscosity, erythrocytes resistance
osmotic resistance
5
Medical Biology Data about osmosis, osmotic
pressure
Pathophysiology About metabolic and Assess given indexes of acid-
respiratory alkalosis and alkaline equilibrium and make a
acidosis reasons and conclusion about possible reasons
developmental mechanisms and developmental mechanisms

3.2.Topic content
INTRODUCTION
Body is formed by solids and fluids. The fluid part is more than 2/3 of the whole body.
Water forms most of the fluid part of the body.
In human beings, the total body water varies from 45 to 75% of body weight. In a normal
young adult male, body contains 60-65% of water and 35-40% of solids. In a normal young
adult female, the water is 50 to 55% and solids are 45-50%. In females, water is less
because of more amount of subcutaneous adipose tissue. In thin persons, water content is
more than in obese persons. In old age, water content is decreased due to increase in
adipose tissue. The total quantity of body water in an average human being weighing about
70 kg is about 40 l iters.
1.. Isotonic Solutions
The solutions having the same effective osmolality (tonicity) as body fluids are
called isotonic solution. The examples are 0.9% sodium chloride solution (normal
saline) and 5% glucose solution.
The red blood cells placed in isotonic solution (normal saline) neither gain nor lose
water by osmosis (Fig. 1). This is because of the osmotic equilibrium between inside and
outside the cell across the cell membrane.
2.. Hypertonic Solutions
Solutions like 2% sodium chloride solution, having greater effective osmolality than
the body fluids are called hypertonic solutions. When red blood cells are placed in
hypertonic solution, water moves out of the cells (exosmosis) resulting in shrinkage of
the cells (crenation).

6
 FIGURE 1: Effect of hypertonic and hypotonic solutions on red blood cells

3. Hypotonic Solutions
The solutions, which have less effective osmolality than the body fluids are called
hypotonic solutions. The example is 0.3% sodium chloride solution. When the red
blood cells are taken in hypotonic solution, water moves into the cells (endosmosis)
resulting in swelling and rupture (hemolysis) of the cells.
APPLIED PHYSIOLOGY—DEHYDRATION
DEFINITION
Significant decrease in water content of the body is known as dehydration. This is
observed more commonly in children than adults.
CONDITIONS WHEN DEHYDRATION OCCURS
Dehydration is common in the following conditions:
1. Severe diarrhea and vomiting leading to decrease in body water, sodium and other
electrolytes.
2. Increased urinary output because of renal diseases or adrenal insufficiency leading
to lack of aldosterone. In the absence of aldosterone, the reabsorption of sodium and
chloride by the renal tubules reduces and urine output increases.
3. Insufficient intake of water.
4. Excessive sweating leading to heat frostration, i.e. extreme loss of water, heat and
energy.
COMPLICATIONS OF DEHYDRATION
Dehydration reduces blood volume and cardiac output resulting hypovolemic
cardiac shock. In severe conditions of dehydration, renal failure and coma occur.
Blood is a connective tissue in fluid form. It is considered as the fluid of life because it
carries oxygen from lungs to all parts of the body and carbon dioxide from all parts of
the body to the lungs. It is known as fluid of growth because it carries nutritive
substances from the digestive system and hormones from endocrine gland to all the
tissues. The blood can also be called the fluid of health. Because it protects the body

7
against the diseases and gets rid of the waste products and unwanted substances by
transporting them to the excretory organs like kidneys.

PROPERTIES OF BLOOD
Following are the properties of the blood.
1. Color:
2. Volume: The volume of blood in a normal adult is 5 liters.
3. Reaction and pH: Blood is slightly alkaline and its pH in normal conditions is 7.4.
4. Specific gravity:
The specific gravity of total blood: 1.052 to 1.061
The specific gravity blood cells: 1.092 to 1.101
The specific gravity of plasma: 1.022 to 1.026
5. Viscosity: Blood is five times more viscous than water.
It is mainly due to red blood cells and plasma proteins.
6. Blood temperature
7. Blood densit: 1,056-1,060.

Blood color - depends on hemoglobin. Blood is red in color. Arterial blood is brightly
red because of oxyhemoglobin (hemoglobin connected with oxygen) big amount.
Venous blood is dark red with blue shade or purple red. Such blood color is linked with
presence not only of oxydated hemoglobin but also reducted hemoglobin.
Blood temperature depends greatly on organ metabolism level from which it
outflows. The more intensive metabolism level is, the higher is the temperature of
blood that outflows from it. Thus, venous blood temperature is bigger always in one
and the same organ than the one of the arterial blood. Though, this rule does not
correspond to skin superficial veins because they are in touch with atmospheric air and
participate indirectly in heat-exchange. Blood temperature is fluctuated from 37 to
40°C under resting conditions in different vessels in homoiothermal animals and
human being. So, blood outflowing from lover through veins can have the temperature
equal to 39,7°C. Blood temperature is increased significantly at intensive muscular
activity.
Blood density is increased at blood condensation; is decreased – at its liquerfaction.
Density level depends on formed elements (mainly erythrocytes) amount and proteins
concentration.
Acid-alkaline equilibrium – correlation between acid and alkaline equivalent in
blood. This is reaction caused by H+-ions concentration. Ph or hydrogen index is used
for its evaluation. If pH is equal to 7,0 the environment is called neutral; less than 7,0
– acid; more than 7,0 – alkaline. Norma: in venous blood – 7,34, arterial blood – 7,4;
blood in a whole – 7,35-7,47. At muscular tension increasing acid products come into
blood (lactic acid, carbonic acid) and movement to acid side (acidosis) is observed. At
increased carbonic acid releasing (with lungs at hyperventillation) movement to
alkaline side (alkalosis) occurs.
COMPOSITION OF BLOOD
Blood contains the blood cells which are called formed elements and the liquid
portion known as plasma.
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 BLOOD CELLS
Three types of cells are present in the blood.
1. Red blood cells or Erythrocytes
2. White blood cells or Leukocytes
3.Platelets or Thrombocytes
Hematocrit Value
If blood is collected in a hematocrit tube along with suitable anticoagulant and
centrifuged for 30 minutes with a speed of 3000 RPM (revolutions per minute), there are
blood cells settle down at the bottom having a clear plasma at the top. The plasma forms 55%
and the red blood cells form 45% of the total blood. The volume of red blood cells
expressed in percentage is called the hematocit value or packed cell volume (PCV).
In between the plasma and the red blood cells there is a thin layer of white buffy coat.
This white buffy coat is formed by the aggregation of white blood cells and platelets.

PLASMA
Plasma is a straw coloured clear liquid. It contains 91 to 92% of water and 8 to 9%
of solids. The solids are the organic and the inorganic substances.

Organic Substances of the Plasma

The following are the organic substances of the plasma.
1. Proteins: The proteins present in the plasma are albumins, globulins and
fibrinogen. These are specifically known as plasma proteins.
2. Carbohydrates: The carbohydrate is present in plasma mainly in the form of
glucose.
3. Fats: The lipid substances present in plasma are the neutral fats, phospholipids
and cholesterol.
4. Amino acids: Plasma contains both essential and nonessential amino acids.
5. Nonprotein nitrogenous substances: The plasma also contains some nonprotein
nitrogenous substances like ammonia, creatine, creatinine, xanthine, hypoxanthine,
urea and uric acid.
6. Internal secrets: The plasma contains many hormones.
7. Enzymes: The enzymes like amylase, carbonic anhydrase, alkaline phosphatase,
acid phosphatase, lipase, esterase, protease and transaminase are present in plasma.
8. Antibodies: The plasma contains many antibodies, which are called
immunoglobulins.
Inorganic Substances of the Plasma
Following are the inorganic substances present in plasma.
1. Sodium
2. Calcium
3. Potassium
4. Magnesium
5. Chloride
6. Iodide
7. Iron
8. Phosphates
9
 9. Copper.
Gases Present in Plasma
Oxygen and carbon dioxide are also present in plasma.
The Table 1 gives the normal values of some important substances in blood.
TABLE 1: Blood level of some important substances
Glucose 3,5-5,5 mmol/l
Cholesterol 3,9-6,5 mmol/l
Plasma proteins (globulins, albumins, fibrinogen) 23-35 g/l, 35-50 g/l, 2-4 g/l
Iron 11-31 mcmol/l
Calcium 2,25-2,74 mmol/l
Bilirubin (total, unconjugated, conjugated) 8,55-20,52, 1,7-17,1, 2,2-5,1
mcmol/l
FUNCTIONS OF BLOOD
1. NUTRIENT FUNCTION
Nutritive substances like glucose, amino acids, lipids and vitamins derived from
digested food are absorbed from gastrointestinal tract and carried by blood to different
parts of the body for growth and for production of energy.
2. RESPIRATORY FUNCTION
Transport of respiratory gases is done by the blood. It carries oxygen from alveoli
of lungs to different tissues and carbon dioxide from tissues to alveoli.
3. EXCRETORY FUNCTION
Waste products formed during various metabolic activities the tissues are removed
by blood and carried to the excretory organs like kidney, skin, liver, etc.
4. TRANSPORT OF HORMONES AND ENZYMES
The hormones and some of the enzymes are carried by blood to different parts of
the body from the source of secretion.
5. EGULATION OF WATER BALANCE
Water content of the blood is freely interchangeable with interstitial fluid. This helps
in the regulation of water content of the body.
6. REGULATION OF ACID-BASE BALANCE
The plasma proteins and hemoglobin act as buffers and help in regulation of acid
base balance.
7. REGULATION OF BODY TEMPERATURE
Because of the high specific heat of blood, it is responsible for maintaining the ther-
moregulatory mechanism in the body, i.e. the balance between heat loss and heat gain
in the body. Not only temperature making equal but also creating conditions for heat-
production or heat-releasing in organism occurs at blood movement. Bigger blood
amount passage through skin vessels in hot weather encourages to heat-emission.
Cutaneous vessels are constricted in cold weather, blood is pushed in abdominal cavity
that leads to heat-saving.
8. STORAGE FUNCTION
Water and some important substances like proteins, glucose, sodium and
potassium are оnstantly required by the tissues. Blood serves as a readymade source for

10
these substances. And, these substances are taken from blood during the conditions like
starvation, fluid loss, electrolyte loss, etc.
9. DEFENSIVE FUNCTION
Blood plays important role in the defense of the body. The white blood cells are
responsible for this function. Neutrophils and monocytes engulf the bacteria by
phagocytosis. Lymphocytes are involved in immunity (both humoral and cell-mediated).
Eosinophils are responsible for detoxification, disintegration am removal of foreign
proteins. In general, blood coagulation and fibrinolysis (clot destruction), antioxidants
belong to this wide function.
The proteins in the plasma are:
1. Serum albumins
2. Serum globulins and
3. Fibrinogen.
When the blood is shed out of the blood vessel or collected in a container, the
coagulation occurs. In this process, the fibrinogen is converted into fibrin and the
blood cells are attached to this fibrin forming the blood clot. After about 45 minutes,
a straw colored fluid leaves blood clot. This fluid is called serum. The serum is different
from plasma in its protein content. The serum contains only the albumins and globulins.
The fibrinogen is absent in serum because, it is converted into fibrin during the
coagulation of the blood. Serum contains all the other constituents of plasma except
fibrinogen.
Thus, the Serum = Plasma - Fibrinogen. Because of this, the albumins and
globulins are usually called serum albumins and serum globulins.
NORMAL VALUES
The normal values of the plasma proteins are:
Total proteins : 60-80 g/l
Serum albumins : 35-50 g/l,
Serum globulins : 23-35 g/l
Alpha1 - : 1-4 g/l
Alpha2 - : 4-12 g/l
Beta- : 5-11 g/l
Gamma- : 5-16 g/l
Fibrinogen : 2-4 g/l

PROPERTIES OF PLASMA PROTEINS

The following are the properties of plasma proteins.
1. MOLECULAR WEIGHT
The molecular weight of albumin : 69,000
The molecular weight of globulin: 156,000
The molecular weight of fibrinogen: 4,00,000
Thus, the molecular weight of fibrinogen is greater than that of other two proteins.
2. ONCOTIC PRESSURE
The plasma proteins are responsible for the oncotic or osmotic pressure in the blood.
The osmotic pressure exerted by proteins in the plasma is called colloidal osmotic
pressure. Oncotic pressure represents osmotic pressure part and depends on proteins
11
content in solution. Although proteins concentration is rather big in a solution, molecules
general amount is relatively low due to their big molecular weight. That is why oncotic
pressure is not more than 25-30 mm Hg. Albumins play a major role in exerting oncotic
pressure. The share of their contribution to oncotic pressure determining is 80%. It is so
because of their low molecular weight and molecules big amount in plasma.
Oncotic pressure plays important role in watery exchange regulation. The bigger is
oncotic pressure, the more water is maintained in vascular bed and the less is transported
into the tissues and on the contrary. Oncotic pressure not only influences on tissular
liquid and lymph formation but also regulates uroformation and water absorbtion in
intestine.
Hypoproteinemy observed at proteinic fasting as well as kidneys hard pathological
conditions leads to edemas because water can not be maintained in vascular bed and
comes to the tissues.
3. SPECIFIC GRAVITY
The specific gravity of the plasma proteins is 1,026.
4. BUFFER ACTION
The acceptance of hydrogen ions is called buffer action. At birth acidosis is
physiologic. Ph is supported by: buffer systems, excretory organs and lungs.
Buffer systems:
• bicarbonate,
• phosphate,
• protein,
• hemoglobin (75 per cent of all system).
The plasma proteins have 1/6 of total buffering action of the blood.

ORIGIN OF PLASMA PROTEINS

IN EMBRYO
In embryonic stage, the plasma proteins are synthesized by the mesenchyme cells. In
embryo, the albumins are synthesized first and other proteins are synthesized later.
IN ADULTS
In adults, the plasma proteins are synthesized mostly from reticuloendothelial cells of liver.
The plasma proteins are also synthesized from spleen, bone marrow and disintegrating
blood cells and general tissue cells. Gamma globulin is synthesized from B-
lymphocytes.

FUNCTIONS OF PLASMA PROTEINS

The plasma proteins are very essential for the body. The following are the various
functions of the plasma proteins
1. ROLE IN COAGULATION OF BLOOD
Fibrinogen is essential for the coagulation of blood. During coagulation of blood, the
fibrinogen is converted into fibrin.
2. ROLE IN DEFENSE MECHANISM OF BODY

12
 The gamma globulins play an important role in the defense mechanism of the body by
acting as antibodies (immune substances). These protein are also called immunoglobulins.
The antibodies react with antigens of various microorganisms, which cause diseases like
diphtheria, typhoid, streptococcal infections, mumps, influenza, measles, hepatitis,
rubella, poliomyelitis, etc.
3. ROLE IN TRANSPORT MECHANISM
Plasma proteins are essential for the transport of various substances in the blood.
Albumin, alpha globulin and beta globulin are responsible for the transport of the hormones,
enzymes and respiratory gases, particularly carbon dioxide. The alpha and beta
globulins play an important role in the transport of metals in the blood.
4. ROLE IN MAINTENANCE OF ONCOTIC PRESSURE IN BLOOD
Because of their large size, the plasma proteins cannot pass through the capillary
membrane easily and remain in the blood. In the blood these proteins exert the colloidal
oncotic pressure. The oncotic pressure exerted by the plasma proteins is about 25 mm
Hg. In this way, the plasma proteins play an important role in the maintenance of oncotic
pressure of blood.
Since the concentration of albumins is more than the other plasma proteins, it exerts
maximum pressure, globulins are the next and fibrinogen exerts least pressure.

Importance of Oncotic Pressure - Starling's Hypothesis

Oncotic pressure exerted by the plasma proteins is involved in the exchange of
various substances between blood and the cells through capillary membrane. Accord-
ing to Starling's hypothesis, the net filtration through capillary membrane is
proportional to the hydrostatic pressure difference across the membrane minus the
oncotic pressure difference.
5. ROLE IN REGULATION OF ACID-BASE BALANCE
Plasma proteins, particularly the albumins, play an important role in regulating the
acid- base balance in the blood. This is because of the virtue of their buffering action
6. ROLE IN VISCOSITY OR INTERNAL FRICTION OF BLOOD
It is often determined comparatively to water viscosity: if the latest one is equal to
1, blood viscosity is equal to 4,0-5,0. In new-borned - 10,0-14,0; in 1 month – like in
the adult. In girls this index is less than in boys. Main reasons for viscosity increasing:
• in mountains;
• hypercapnia (carbonic acid content increasing in blood);
• inflammations;
• hypertony;
• atherosclerosis;
• at feeding of mainly animal food (proteins-rich) – meat, eggs.

Main reasons for viscosity decreasing:

• at vegeteranian feeding.
Viscosity depends on formed elements (mainly erythrocytes) amount and proteins
concentration. The plasma proteins provide viscosity to the blood, which is important

13
to maintain the blood pressure. Albumin provides maximum viscosity than the
other plasma proteins.
7. ROLE IN ERYTHROCYTE SEDIMENTATION RATE (ESR) EITHER
BLOOD SUSPENSIONAL STABILITY OR VELOCITY SEDIMENTATION
RATE (VSR).
Blood represents suspension from physical-chemical point of view because formed
elements are in plasma in a suspended state. Suspension is a liquid containing other
substance particles that are distributed equally. Erythrocytes suspension in plasma is
sustained by their surface hydrophilic nature as well as their negative membrane
surface charge that they possess like other formed elements. Thus, erythrocytes are
pushed away one from another. If formed elements negative charge is reduced that can
be connected with positive proteins of cathions adsorbtion than favorable conditions
are created for erythrocytes mutual gluing. Such a gluing is especially powerful at
fibrinogen and gamma-globulins adsorbtion on their membrane. Mentioned proteins
form so-called bridges between separate erythrocytes due to which their aggregation
occurs and so-called coin columns are formed. Real power of aggregation represents
difference between force in formed bridges, force of negatively charged red blood cells
electrostatic pushing away and shift power causing aggregates decomposition. It is not
excluded that proteins molecules connection at the erythrocytes surface takes place due
to weak hydrogenic bonds as well as dispersal forces of Van-der-Waals.
If erythrocytes aggregation is observed in organism than blood viscosity is increased
that can create favorable conditions for blood intravascular coagulation as well as blood
pressure increasing. Moreover, “coin columns” prevent cells, tissues and organs
normal blood supply at their sludging in capillaries.
If one puts blood in the test-tube after preliminary adding substances preventing
coagulation than one can see after some time that plasma was divided into two layers:
the superior one consists mainly of plasma and the inferior one – of formed elements.
Taking into account mentioned features Ferreus proposed to study erythrocytes
suspension stability while their sedimentation determining in blood the coagulation of
which is liquidated by preliminary adding of citric sodium. Nowadays this reaction is
known as erythrocytes sedimentation velocity or rate (ESR). Other name is velocity
sedimentation rate (VSR). This method is rather simple in usage, non-specific (gives
information at many physiological and pathological conditions) and that is why is still
judiciously used in theoretical and practical medicine all over the world.
Normal VSR is determined by plasma normal proteinogram. This index depends on
age and sex. It is equal to 6-12 mm/h in adult men, 8-15 mm/h in adult women, 15-20
mm/h in the old of both sexes, 1-2 mm/h in babies (it reaches adult values up to sexual
maturation period). The biggest contribution to VSR has highly-molecular protein
fibrinogen. Women have bigger fibrinogen level than men (it is physiological facility
to prevent regular menstrual bleedings and bleedings during labors). That is why VSR
can reach even 15-20 mm/h at menstruations and 20-30 mm/h during pregnancy.
Erythrocytes concentration and proteins (especially highly-molecular ones) content
are two distinguishing factors that determine VSR level. So, if erythrocytes number is
little (anemia) than the resting number of them are easily to be sedimentated than to
their normal or increased number in one and the same volume. Thus, anemia is
14
accompanied by increased VSR, erythrocytosis (red blood cells increased number) –
decreased VSR.
Inflammatory, infection diseases are accompanied by proteins content increasing in
blood (immunoglobulins at infection, acute-phased proteins at inflammation)
especially of highly-molecular ones. It leads to VSR rising. Tumors (especially
cancerogenous) are connected with very-highly molecular proteins production which
are absorbed on cells (erythrocytes in part). As the scientists concluded, VSR rising
from 40 and upper can testify to tumors or temporal arteriitis (this disease should be
excluded). VSR equal to 100 mm/h is a cancer distinguishing feature. Burnings and
combustions are accompanied by tissular proteins enforced decomposition
(catabolism) and, thus, hyperproteinemy, that leads to VSR rising. Other pathological
conditions the VSR increasing at which is a diagnostic criterium are as follows as:
myocardial infarction, post-operative period, hemoblastoses (myelomic disease at
which Bens-Jons' protein causes occlusion in vessels in kidney capillaries in part that
can lead to kidney insufficiency and death; Waldenstrem's disease, disease of
immunoglobulins hard chains at which lymphocyte anomalous precursor produce only
hard chains of lg that lead to their level rising in blood and thus enforced absorbtion
onto erythrocytes external membrane), chronic active hepatitis (as a result of
hyperglobulinemy), liver cirrhosis, tuberculosis, amyloidosis, collagenoses (tissues
proteins catabolism).
VSR reducing is observed at erythremy and symptomatic erythrocytoses
(erythrocyte number increasing), viral hepatitis (hypoglobulinemy result), mechanic
jaundice, different hypoproteinemies, salicylates and calcium chloride taking. VSR
decreasing lower than 3 mm/l is unfavorable diagnostic sign because it testifies to
blood viscosity increasing.
VSR level depends more on plasma features in bigger extent than on erythrocytes
ones. So, if to put male erythrocytes with normal VSR in a pregnant woman plasma
than they will begin their sedimentation with the same velocity like female erythrocytes
during pregnancy.
8. ROLE AS RESERVE PROTEINS
During the conditions like fasting, inadequate food intake or inadequate protein
intake, the plasma proteins are utilized by the body tissues. Because of this, the plasma
proteins are called the reserve proteins.
Shortly,
Albumins – (60 % or 35-50 g/l) produced in liver; are agile, low-weighted, they are
important for:
• oncotic pressure support;
• bilirubin transport;
• hard metals salts transport;
• fat acids transport;
• medicines transport (proteins increase action periods for them).
Globulins (40% or 30-35 g/l) are formed in liver, bone marrow, spleen. Role:
• form antibodies;
• antitoxins;

15
• agglutinines;
• blood coagulation (some of them are clotting factors);
• phospholipid transport;
• cholesterol transport;
• steroid hormones transport;
• oncotic pressure support (less than albumins);
• blood density support;
• buffers;
• blood viscosity determination;
• nutritive function.
VARIATIONS IN PLASMA PROTEIN LEVEL
The level of plasma proteins vary independently of one another. However, in several
conditions, the quantity of albumin and globulin change in opposite direction.
DECREASE IN ALL FRACTIONS OF PROTEINS–HYPOPROTEINEMIA
Hypoproteinemia occurs in the following conditions:
1. Hemorrhage
2. Extensive burns
3. Pregnancy
4. Malnutrition
5. Prolonged starvation
6. Cirrhosis of liver and
7. Chronic infections like chronic hepatitis or chronic nephritis.
INCREASE IN ALL FRACTIONS
HYPERPROTEINEMIA
Hyperproteinemia occurs in the following conditions:
1. Dehydration
2. Acute infections like acute hepatitis or acute nephritis.
1. Materials for auditory self-work.

4.1. List of study practical tasks necessary to perform at the practical class.
Materials and methods: scarificator, pipettes, blood dilutor, Panchenkov’s device,
test tubes, centrifuge, blood.
Investigation object: rat.
Task 1. To get acquainted with blood taking technology for analysis
performance.
The investigator must wipe investigated person left fourth finger with cotton wool
washed in alcohol. To prick the cured finger with sterile scarificator. To dry first blood
drop with cotton wool. To put the end of horizontally located pipette in the second
blood drop and to fetch very carefully (without vesicles) blood till corresponding mark.
To gain this goal it’s necessary to immerse the pipette in complete blood drop. It’s
necessary to dry pipette end with cotton wool and to perform manipulations with blood
according to investigation character.

Task 2. To determine erythrocytes osmotic resistance.

16
 To put 20 test tubes in support and to number them. To pour hypotonic solutions in
the dosage of 1 ml in corresponding test tubes (concentrations from 0,60 to 0,15 per
cent with the difference in 0,05 per cent in every test tube). To add investigated blood
in every test tube. To mix carefully the content and to stay them at room temperature
for 15 minutes. To centrifugate after this at 1500 rotations per minute in course of 5
minutes. To determine the limits of erythrocytes maximal and minimal resistance.
Erythrocytic resistance - is red blood cells feature to resist injured actions (osmotic,
chemical, mechanical and so on). But osmotic resistance to sodium chloride hypotonic
solutions is the most-spread resistance index determined under clinical conditions (it is
rather easily to be performed and to evaluated comparatively to other resistance types).
Under norm minimal resistance (hemolysis beginning) – at sodium chloride content
0,42-0,48 per cent; maximal resistance (complete hemolysis) – at 0,30-0,34 per cent of
it. In a fresh blood- 0,20-0,40 per cent of NaCl; in incubated one (in course of 24 hours)
– 0,20-0,65 per cent of NaCl.
It is decreased at:
• congenital microspherocytic hemolytical jaundice;
• new-borns haemolytical disease;
• toxicoses;
• acute infections;
• leukemias;
• lymphogranulematoses;
• hepatic cirrhoses;
• ABO- and Rh- blood incompatibility.
It is increased at:
• drepanocytic anaemias;
• mechanical jaundices.
But this index is the most significant at anemias differentiated diagnosis.

Task 3. Velocity sedimentation rate (VSR) determining.

To wash capillary pipette of Panchenkov with 5% solution of citrate sodium. To
fetch this solution till the mark 75/25 mm3 and to blow it to the clock glass.
To prepare the finger, to prick it and to fetch blood till the mark 100 mm3. To blow
blood to the clock glass and to mix it with citrate sodium in correlation of 1:4. To fill
the pipette with this citric blood exactly till the mark “K” and to put it into support
vertically for 1 hour. In 1 hour to determine the highland in mm of plasma column
above formed elements.

2. Literature recommended:
1. Lecture course.
2. Mistchenko V.P., Tkachenko E.V. Methodical instructions for dental students
(short lecture course).-Poltava, 2005.-P.38-39.
3. Mistchenko V.P., Tkachenko E.V. Methodical instructions for medical students
(short lecture course).-Poltava, 2005.-P. 60-62.

17
 4. Mistchenko V.P., Tkachenko E.V. Blood system Physiology //Methodical
recommendations to practical classes for students of medical and dental
departments.-Poltava, 2005.-20 p.
5. Kapit W., Macey R.I., Meisami E. The Physiology Colouring Book: Harpers
Collins Publishers, 1987.-P. 135.
6. Guyton – Ganong – Chatterjee. Concise Physiology /Ed. By Dr Raja Shahzad
Gull: M.B.B.S., F.C.P.S., King Edward Medical College.-Lahore, 1998 (1st
Edition).-P.170-171, 203-204.
7. Stuart Ira Fox. Human Physiology.-8th Ed.-McGrawHill, 2004.-P.367-368, 377-
378.
8. Seeley R.R., Stephens T.D., Tate P. Essentials of Anatomy and Physiology.-The
3rd Ed.-McGraw Hill, 1999.-P.286-288.
2. Materials for self-control:
Control questions:
1. Blood system.
2. Blood content, its amount.
3. Blood functions.
4. Blood constants and their significance in clinical practice.
5. Blood main components, hematocrit.
6. Blood buffer systems, acidosis, alkalosis.
7. Erythrocytic osmotic resistance.
8. Velocity sedimentation rate, factors, influencing on it, diagnostic value.
9. Osmotic pressure.
10. Oncotic pressure.
11. Blood viscosity.
12. Blood temperature, blood color, factors they depend on.

LESSON 32
ERYTHROCYTES NUMBER AND HEMOGLOBIN CONCENTRATION
INVESTIGATION

1. The topic studied actuality.

Erythrocytic stability to hemolysis is decreased at some diseases. Erythrocytes
indexes determining is widely used all over the world for anemias differential
diagnostics. Dentists can deal with anemias connected with salivary glands (for
instance, parotid) pathology.
Erythrocytes morpho-functional indexes can be changed at oral cavity diseases in
part of salivary glands, at phlegmons and abscesses.
2. Study aims:

18
 To know: erythrocytes and hemoglobin structure, functions and normal value,
representation about color index, erythrocytes hemolysis and influenced factors as well
as erythropoiesis and its regulation.
To be able to: count Er and Hb level in blood as well as color index.

Pre-auditory self-work materials.

3.1.Basic knowledge, skills, experiences, necessary for study the topic:

Subject To know To be able to
Medical biological Data about hemoglobin
chemistry structure
Biophysics Data about erythrocytes Assess osmotic hemolysis of
hemolysis erythrocytes
Medical Biology Data about microscope Work with microscope, investigate
main structural parts hyperosmotic and hypoosmotic state in
sodium chloride different-concentrated
solutions
Histology, Cytology Data about microscope Work with microscope, recognize
and Embryology main structural parts, preparations of erythropoiesis at its
representations about different stages, tell about
erythropoiesis and its erythropoiesis on Chertkov-Vorobiyev
regulation scheme
Pathophysiology About anemias reasons, Assess anemias types on the base of
types, main given indexes of erythrocytes number,
developmental hemoglobin concentration, colour
mechanisms index

3.2. Topic content

It is interesting to know
• If to put one erythrocyte on another one than one can receive the “column” more
than 60 km in height.
• All erythrocytes surface is equal to 4000 m2 in one human being.
• To count all erythrocytes in one human being one needs 475000 years if to count
them with the velocity equal to 100 red blood cells in 1 min.
• There exists plant hemoglobin – legoglobin or leghemoglobin. It is present in the
legumes (beans). Hem is produced by plant, while proteinic part – by bacterias that
live on these plants and convert atmospheric nitrogen to nitrogen-containing
fertilizations. It is an example of double symbiosis in alive nature.
• Current research is being conducted in an attempt to develop artificial hemoglobin.
One chemical that has been used in clinical trials is a perfluorochemical emulsion
called Fluosol DA, a white liquid with a high oxygen affinity. Although the
usefulness of hemoglobin substitutes is currently limited because artificial
hemoglobin is destroyed fairly quickly in the body, future work may uncover more
successful substitutes that can provide long-term relief for patients with blood

19
 disorders. The use of artificial hemoglobin could eliminate some of the
disadvantages of using blood for transfusions. With artificial hemoglobin,
transfusion reactions would not occur because of mismatched blood, and
transferring diseases such as hepatitis or AIDS would be eliminated. In addition,
artificial hemoglobin could be used when blood is not available.
INTRODUCTION AND NORMAL VALUE
Erythrocytes or red blood cells (RBC) are the non-nucleated formed elements in the
blood. The red colour of these cells is due to the presence of the colouring matter-
hemoglobin in these cells. The word “erythros” means red.
Normal Values:
• in adult men – 4,5-5,5 x 1012/l;
• in adult women – 3,7-4,5 x 1012/l;
• in newborns – up to 7,0 x 10 12/l;
• adult ciphras – up to adolescence.
MORPHOLOGY OF RED BLOOD CELLS
NORMAL SIZE
• diameter – 7,0-7,7 mcm – in normocytes; less than 6,0 mcm – microcytes, more
than 7,7 mcm macrocytes;
• width – 2 mcm;
• volume – 76-100 mcm;
• surface square – 140-150 mcm2

2.2 p
FIGURE 2: Dimensions of red blood cell. A: Surface view. B. Sectioned view
Normally, the red blood cells are disc shaped and biconcave (dumb-bell shaped). The
biconcave contour of red blood cells has the following mechanical advantages.

20
 1. It helps in equal and rapid diffusion of oxygen and other substances into the
interior of the cell.
2. Large surface area is provided for absorption or removal of different substances.
3. Minimal tension is offered on the membrane when the volume of cell alters.
4. While passing through minute capillaries, these cells can squeeze through the
capillaries very easily.
PROPERTIES OF RED BLOOD CELLS
1. ROULEAUX FORMATION
When blood is taken out of the blood vessel, the red blood cells pile up one above another
like the pile of coins. This property of the red blood cells is called rouleaux (pleural =
rouleau) formation (Fig.3).
2. SPECIFIC GRAVITY
The specific gravity of red blood cell is 1.092 to 1.101.
3. PACKED CELL VOLUME
When the blood is collected in a centrifuge tube along with proper anticoagulant
and centrifuged for a period of 30 minutes at a speed of 3000 rpm (revolutions per
minute), the red blood cells settle at the bottom of the tube leaving the clear plasma
at the top. The red blood cells form 45% of the total blood. This is called the packed cell
volume or hematocrit. The volume of plasma is 55%.
4. SUSPENSION STABILITY
During circulation, the red blood cells remain suspended uniformly in the blood. This
property of the red blood cells is called the suspension stability or sedimentation absence.

FIGURE 3: Rouleau formation Courtesy: Dr Nivaldo Medieiros

This figure demonstrates “coin columns” formation or erythrocytes sedimentation

that has been described in lesson number 31.
VARIATIONS IN NUMBER OF RED BLOOD CELLS
PHYSIOLOGICAL VARIATIONS
A. Increase in the red blood cell count is known as polycythemia. If it occurs in
physiological conditions, it is called physiological polycythemia. Although some

21
authors determine polycytemy as erythrocytes, leucocytes and platelets amount
increase and the term “erythrocytosis” is used for erythrocytes increase
designation. 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 infants. 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.
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.
B. Decrease in red blood cell count (erythropeny) occurs in the following
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 extracellular fluid volume, increases the plasma volume also resulting in
hemodilution. So, there is a relative reduction in the red blood cell count.
VARIATIONS IN SIZE OF RED BLOOD CELLS
The size of the red blood cells alters in various conditions.
Microcytes
Microcytes are the red blood cells of small size and are present in the following
conditions:
1. Iron deficiency anemia
2. Prolonged forced breathing and
3. Increased osmotic pressure in blood
22
 Macrocytes
Macrocytes are the red blood cells with larger size. The macrocytes are present in
the following conditions:
1. Megaloblastic anemia
2. Muscular exercise and
3. Decreased osmotic pressure in blood
4. Disease of Minkovsky-Shoffar – when erythrocytes are big and spheric
Anisocytes
The red blood cells with unequal size are called the anisocytes. This happens in
pernicious (zyancobalamin-deficient) anemia.
So, one can tell about about 3 states:
• microcytosis;
• macrocytosis;
• anisocytosis.
Erythrocytes size assessment is rather essential at anemias differential diagnostics.
VARIATIONS IN SHAPE OF RED BLOOD CELLS
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. Examples:
• crenation – shrinkage as in hypertonic solution;
• spherocytosis – globular form as in hypotonic solution;
• elliptocytosis – elliptical shape as in certain types of anemia;
• sickle cells or drepanocytes – crescentic shape as in sickle cell anemia;
• planocytes – flattened;
• pear-shaped;
• racket-shaped;
• flask-shaped;
• hammer-shaped;
• stomatocytes – concave from one side;
• echinocytes – “hedgehogs”;
• acantocytes – like the previous ones, but “needles” are wider and less in
amount; as a rule, these change is irreversible one comparatively to the previous
one;
• codocytes – target cells – erythrocytes with a fovea in the center of red cell et
all.
Poikylocytosis – different-shaped erythrocytes presence in one blood smear if
discocytes level is less than 90% while other-shaped erythrocytes percentage is more
than 90%.

LIFESPAN AND FATE OF RED BLOOD CELLS

RBC life duration is 60-120 days (in males – on 10-20 days longer). They are formed
in bone marrow from reticulocytes. At this stage reaching capillary wall is stretched,
vessel is opened and Er are washed into blood stream, where they are transformed into
young Er (normocytes) after 35-45 hours. Er die in liver, spleen. Destructed Er amount

23
corresponds to formed Er amount. This closed system with all mass of Er circulating
into organism received the name erythron.
Average lifespan of red blood cell is about 120 days in men and 110 days in women.
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 cells 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.

DETERMINATION OF LIFESPAN OF R E D B L O O D C E L L
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.
FUNCTIONS OF RED BLOOD CELLS
1. Respiratory – O2 and CO2 transport. Erythrocytes transport oxygen from the lungs
to the tissues. The hemoglobin in red blood cell combines with oxygen and 97% of
oxygen is transported as oxyhemoglobin.Red blood cells transport carbon dioxide
from the tissues to the lungs. The hemoglobin in red blood cell combines with carbon
dioxide and form carbhemoglobin. About 30% of carbon dioxide is transported in this
form.
2. Metabolic – participation in proteins, fats and carbohydrates, water and salts
exchange. When Er passes into capillary arterial end, than it gives water and oxygen
soluble in it id est wrinkles. On the contrary, it swells in venous end because it takes
water, carbonic dioxide and metabolism products. Er help in plasma homeostasis
support. It deals not only to salts but also to proteins. Hyperproteinemy is accompanied
by proteins active absorbtion by Er, hypoproteinemy – proteins giving by Er into
plasma. For example, fibrinogen is adsorbed on Er membrane if its level is more than
1,5 g/l in plasma. If fibrinogen content is diminished (for example, at intensive blood
intravascular coagulation) than Er give fibrinogen into plasma. Er are glucose carriers.
3. Transport - oxygen, carbonic dioxide, aminoacids, proteins, enzymes, hormones,
fats, carbohydrates, cholesterol, medicines, different biologically-active substances
(proglandines, leucotrienes, cytokines et al.), microelements. For instance, aminoacids
are transported by erythrocytes in bigger extent than by plasma.
4. Buffer (hemoglobin buffer).
24
 5. Participation in iron metabolism.
6. Bile-formation regulation and participation in biliary pigments metabolism.
7. Antitoxic function.
8. Participation in vascular-platelet hemostasis, blood coagulation and fibrinolysis.
9. Red blood cells carry the blood group antigens like A agglutinogen, B
agglutinogen and Rh factor. This helps in determination of blood group and blood
transfusion.
10. Er play significant role in organism specific and nonspecific resistance (in part,
antioxidant function, binding lg G and complement).
11. Er are erythropoiesis regulators because they contain erythropoietic factors that
come into bone marrow at Er destruction and encourage Er formation.
ERYTHROPOIESIS is the process by which the origin, development and maturation
of erythrocytes occur. Hemopoiesis is the process which includes origin, development
and maturation of all the blood cells.
SITE OF ERYTHROPOIESIS
IN FETAL LIFE
During embryonic life, the erythropoiesis occurs in three stages.
1) Mesoblastic stage: During the first two months of intrauterine life, the primitive red
blood cells are produced from mesenchyme of yolk sac.
2) Hepatic stage: From third month of intrauterine life, liver is the main organ that
produces red blood cells. Some erythrocytes are also produced from spleen and
lymphoid organs.
3) Myeloid stage: During the last three months of intrauterine life, the red blood cells are
produced from red bone marrow and liver.
IN POSTNATAL LIFE AND IN ADULTS
In newborn babies, growing children and adults, the red blood cells are produced only
from the red bone marrow.
1. Up to the age of 5 to 6 years: The red blood cells are produced in red bone marrow of
all bones.
2. From the 6th year up to the 20th year: The red blood cells are produced by red bone
marrow of long bones and all the membranous (flat) bones.
3. After the age of 20 years: The red blood cells are produced from all membranous
bones like vertebra, sternum, ribs, scapula, iliac bones and skull bones and from the
ends of long bones. After 20 years of age, the shaft of the long bones becomes yellow
bone marrow because of fat deposition and looses the erythropoietic function.
During disorders of bone, the red blood cells are produced in spleen.

25
 FIGURE 4: Stem cells. L-Lymphocyte. R-Red blood cell. N-Neutrophil. B-Basophil. E-
Eosinophil. M-Monocyte. P-Platelet
STAGES OF ERYTHROPOIESIS
The various stages between stem cell and matured red blood cell are as follows (Fig.
5):
1. Proerythroblast
2. Early normoblast
3. Intermediate normoblast
4. Late normoblast
5. Reticulocyte and
6. Matured erythrocyte.

26
 Erythrocyte Platelets Neutrophils Eosinophil Basophil Monocyte Lymphocyte

FIGURE 5: Stages of erythropoiesis. CFU-E = Colony forming unit—Erythrocyte,

CFU-M = Colony forming unit—Megakaryocyte, CFU-GM = Colony forming unit—
Granulocyte/Monocyte.
RETICULOCYTE
This is otherwise known as immature red blood cell. It is slightly larger than matured
red blood cell. The cytoplasm contains the reticular network or reticulum formed by
remnants of disintegrated organelles. Due to the reticular network, the cell is called
reticulocyte. The reticulum of reticulocyte is stained by supravital stain.
In newborn babies, the reticulocyte count is 2 to 6%, i.e. 2 to 6 reticulocytes are
present for every 100 red blood cells. The number of reticulocytes is reduced during
the first week after birth. Later, the reticulocyte count remains constant at or below 1

27
% of red blood cells. The number may increase whenever there is increased production
and release of red blood cells into the circulation.
The reticulocyte is also basophilic due to the presence of remnants of Golgi
apparatus, mitochondria and other organelles of cytoplasm. During this stage, the cells
can enter the capillaries through the capillary membrane from source of production.
The cells enter the blood through the capillary membrane by means of a process called
diapedesis.
MATURED ERYTHROCYTE
Now, the reticular network disappears and the cell becomes the matured red blood
cell. The matured red blood cell is biconcave and it is smaller in size with a diameter
of 7.2 microns. It attains the biconcave shape. It is with hemoglobin and without
nucleus.
It requires seven days for the development of matured red blood cell from
proerythroblast. It takes five days for the development of reticulocyte. The reticuloctye
takes two more days to become the matured red blood cell.

FACTORS NECESSARY FOR ER YT HROPOIESIS

Various substances are necessary for the development and maturation of
erythrocytes. These factors are classified into 3 categories, namely:
a) General factors
b) Maturation factors and
c) Factors necessary for hemoglobin formation.

According to other classification, there are 2 main erythropoiesis ways:

1) specific – only due to erythropoietin action;
2) non-specific – due to:
• vitamins;
• microelements;
• hormones.
Specific and noon-specific regulatory ways belong to erythropoiesis humoral
regulation.
GENERAL FACTORS
Erythropoiesis is influenced by a variety of general factors namely:
1. Erythropoietin
2. Hormones
3. Hemopoietic growth factors
4. Colony stimulating factors and
5. Vitamins
1. Erythropoietin
This is complex polypeptide. Its amount is increased at:
• bleedings;
• low oxygen partial pressure;
• ascent to height (in the mountains);
• muscular activity.

28
 It is also called hemopoietin or erythrocyte stimulating factor. Erythropoietin
belongs to the substances with relatively slow metabolism. Its half-duration life period
in blood is more than 1,5 hours. About 10% of circulating erythropoietin is released
from organism wih urine. Erythropoietin daily excretion with urine comprises 0,9-4,0
Activity Units.
Chemistry
Erythropoietin is a glycoprotein containing syalic acids. Its molecular weight is
46000 Da. It is synthesized in the form of pro-erythropoietin (193 amino acids) without
specific activity. It comes in plasma in unactive state wher under specific enzyme –
erythrogenin – action – is transformed into active erythropoietin (it consists of 168
amino acids).
Source of Secretion
Erythropoietin is secreted by peritubular capillaries of kidneys (juxta-glomerular
apparatus mainly but epitheliocytes as well), uterus, salivary glands (especially
submandibular ones), monocytes-macrophages, liver (during embryogenesis).
Macrophagal erythropoietin is of huge importance in erythropoiesis regulation because
of central position of macrophages-monocytes in bone marrow erythroid insulas.
Macrophage feeds erythroid insula with erythropoietin, ferritin, iron, vitamins and
other substances. Such erythroid insulas begin their presence from the term of
erythropoiesis in yolk sac.
Stimulant for Secretion
Hypoxia is the stimulant for the secretion of erythropoietin. Hypoxy is accompanied
by enzymes activation in kidney structures (they are sensitive to hypoxy).
Phospholipase A2 releasing leads to prostaglandins E1 and E2 increasing, than
adenylatecyclase level rising up and finally cAMP concentration and activity
increasing in kidney peritubular cells producing erythropoieitn. Epinephrin and
norepinephrin also increase cAMP and cGMP level in kidneys.
Actions of Erythropoietin
Erythropoietin causes formation and release of new red blood cells into circulation.
After secretion, it takes 4 to 5 days to show the action. The hormone promotes the
following processes:
1. Production of proerythroblasts from the stem cells in CFU-E of the bone marrow.
2. Development of proerythroblasts into matured red blood cells through the
normoblastic stages—early, intermediate and late normoblasts and reticulocyte.
3. Release of matured erythrocytes into blood through the capillary membrane
from bone marrow. Even some reticulocytes (immature erythrocytes) are released
along with matured red blood cells.
4. It stimulates synthesis of DNA-dependent RNA.
5. Rhibosomal RNA synthesis starts in 15 min after the cell contact with
erythropoietin, DNA – in 2 hors, ferritin-containing proteins – in 4 hours.
6. The result of cellular metabolism change is erythroid cells proliferative and
hemoglobin-synthesizing ability enforcement.
7. Increasing blood stream in vessels surrounding erythropoietic tissue in bone
marrow.
8. Reticulocytes exit increasing from bone marrow sinusoids into blood.
29
 9. Mitosis number increasing in erythroid cells row.
10. One or several mitotic cycle excluding.

It is very important that the specific erythropoietin-binding receptor structure was

deshiphrated by the scientists.

Synthesis regulation
It is realized at the genetic level. The 7th chromosome is responsible for this. Kidney
structure sensitive to hypoxy represents hem-containing protein (hemoprotein) of
peritubular cells binding oxygen molecule. Hemoprotein oxyform inhibits transcription
of genes responsible for erythropoietin at sufficient oxygenation. On the contrary,
hemoprotein deoxyform lacks its oxygen molecule, its affinity to the gene-operator is
decreased and erythropoietin synthesis is activated.
Main regulator of erythropoietin production is oxygen level in blood or, if to be more
exact, blood oxygen availability for tissues depending, in turn, on:
- oxygen level in blood;
- hemoglobin ability to give oxygen;
- tissues increased needs.
Erythropoietin production is stimulated in mountains where pO2 is decreased in
atmospheric air as well as bleeding decreasing blood oxygenous capacity.
Erythropoietin content is 0,01-0,08 IU/ml in a human being plasma. But it can rise
in 1000 and more times at hypoxy. Erythropoetin has 2 forms alpha- and beta- than are
differed only by carbohydrates number and possessing practically equal biological
activity.
2. Hormones:
• Thyroxine - in addition to erythropoietin, thyroxine also forms an important
general factor for erythropoiesis. Thyroxine accelerates the process of
erythropoiesis at many levels. In hyperthyroidism, polycythemia is common.
• Hypophyseal erythropoietical hormone, ACTH, STH - enforce erythropoiesis.
• Suprarenal glands – glucocorticoids, adrenaline - enforce erythropoiesis.
• Parathyroid - parathormone - enforces erythropoiesis.
• Female sexual organs – erythropoiesis weakening.
• Male sexual organs - enforce erythropoiesis.
3. Hemopoietic Growth Factors
Hemopoietic growth factors or growth inducers are the interleukins and stem cell
factor (steel factor). Generally these factors induce the proliferation of pluripotent stem
cells.
Interleukins (IL) are glycoproteins which belong to the cytokines family. The
interleukins involved in erythropoiesis are.interleukin-3 (IL-3), interleukin-6 (IL-6) and
inter-leukin-11 (IL-11). IL-3 is secreted by T lymphocyte. IL-6 is secreted by T
lymphocytes, endothelial cells and macrophages. IL-11 is secreted by osteoblasts.
4. Colony Stimulating Factors
The colony stimulating factors (CSF) cause the formation of colony forming
blastocytes. There are three types of colony stimulating factors.

30
 1) Granulocyte CSF (G-CSF) secreted by monocytes and endothelial cells.
2) Granulocyte-Monocyte CSF (GM-CSF) secreted by monocytes, endothelial cells
and T lymphocytes
3) Monocyte CSF ( M-CSF) secreted by monocytes and endothelial cells.
5. Vitamins
Some vitamins are also necessary for the process of erythropoiesis. The deficiency
of these vitamins cause anemia associated with other disorders. The vitamins, which
are necessary for erythropoiesis are:
Vitamin B: Its deficiency causes anemia and pellagra.
Vitamin C: Its deficiency causes anemia and scurvy.
Vitamin D: Its deficiency causes anemia and rickets.
MATURATION FACTORS
Vitamin B12, intrinsic factor and folic acid are necessary for the maturation of red
blood cells.
1. Vitamin B12 (Cyanocobalamin)
This is essential for maturation of erythrocytes. The deficiency of vitamin B12 causes
pernicious anemia. So, Vitamin B12 is called antipernicious factor.
Source of Vitamin B12
Vitamin B12 is called extrinsic factor because it is obtained mostly from diet. Its
absorption from the intestine requires the presence of intrinsic factor of Castle. Vitamin
B12 is stored in the liver and muscle (mostly liver). Where necessary, it is transported to
the bone marrow to promote maturation of red blood cells. It is also produced in the large
intestine by the intestinal flora.
Action of Vitamin B12
Vitamin B12 is essential for synthesis of DNA. Its deficit leads to failure in maturation
of the cell and reduction of the cell division. Also, the cells are larger with fragile and,
weak cell membrane.
2. Intrinsic Factor of Castle
This is produced in gastric mucosa. This is essential for the absorption of vitamin B12
from intestine into the blood. In the absence of intrinsic factor, vitamin B12 is not absorbed.
This happens in severe gastritis, ulcers and gastrectomy. The deficiency of intrinsic factor
also causes pernicious anemia since the vitamin B12 is not absorbed. The extrinsic and
intrinsic factors are together call Hematinic principle.
3. Folic Acid
This is also essential for maturation. This is required for the synthesis of DNA. In the
absence of folic acid, the synthesis of DNA is reduced causing failure of maturation.
This leads to anemia in which the cells are large and appear in megaloblastic
(proerythroblastic) stage. And the anemia due to the folic acid deficiency is called
megaloblastic anemia.
FACTORS NECESSARY FOR HEMOGLOBIN FORMATION
Various materials are essential for the formation of globin in the red blood cells.
The deficiency of the substances decreases the production of hemoglobin leading to
anemia.
These factors are as follows:

31
 First class proteins and amino acids: Proteins of highly biological value are essential for
the formation of hemoglobin. Amino acids derived from these proteins are required for the
synthesis of protein part of hemoglobin, the globin.
Iron: Iron is necessary for the formation of heme part of the hemoglobin.
• Copper: This is necessary for the absorption of iron from the gastrointestinal tract.
when copper absence RBC maturate only till reticulocytic stage.
Cobalt and nickel: Cobalt and nickel are essential for the utilization of iron during
hemoglobin formation.
Vitamins: Vitamin C, riboflavin, nicotinic acid and pyridoxine are also essential for
the formation of hemoglobin.

Vitamins action can be described as follows:

Vitamins: of B-group are the most essential:
• B12 (cyancobalamine) - haemopoiesis factor. It is synthesized by microorganisms,
ray fungi and some weads. Cobalt is essential for cyancobalamine formation. This
vitamin comes into human organism with liver, meat, eggs. This vitamin takes part
in haemoglobin synthesis. It is accumulated into liver; its depot is very large (for 5-
10 years).
• B9 (folic acid) – is contained in plant food, liver, eggs. It participates in globin
synthesis influencing on erythroblasts.
• B6 (pyridoxine) catalyzes folic acid formation and cyancobalamine action.
• B2 (rhibophlavine) participates in iron consumption, it also is necessary for
hemoglobin synthesis.
• C (ascorbic acid) – encourages iron releasing from intestine and regulates
hemoglobin synthesis.
• A (retinol) and E (tocopherol) – influence on hemopoietic tissue functions, protect
Er membrane from free radicals action.

Neural-humoral erythropoiesis regulation

It is of less importance than the humoral one. But it is well-known than some
hypothalamic nuclei can stimulate or inhibit erythropoiesis. All these influencings
performance is realized through vegetative nerves. Sympathetic nervous system
excitement is accompanied by erythropoiesis activation. That’s why active life position
and positive emotions - are important erythropoiesis activators.

Erythropoiesis inhibitors
• Fluorum is erythropoiesis inhibitor, that’s why anemia may be developed at its
excess in environment (water, air, foods).
• Estrogens.
• GATA – its absence inhibits completely erythropoiesis.
• NFE-2 – its deficiency disturbs iron absorbtion in intestine and Hb biosynthesis.
Inhibitors are present in blood, they are produced in kidney and liver. Their
action is in following:
❖ hemoglobin synthesis inhibiting;

32
 ❖ prolonging term of young Er one forms transition to the others.

HEMOGLOBIN is the colouring matter of red blood cell. It is a chromoprotein

forming 95% of dry weight of red blood cell and 30 to 34% of wet weight. The function
of hemoglobin is to carry the respiratory gases, oxygen and carbon dioxide. The
molecular weight of hemoglobin is 68,000.
Normal values:
• adult men – 130-180 g/l
• adult women – 120-160 g/l
• newborns – up to 220-240 g/l
DERIVATIVES OF HEMOGLOBIN
Hemoglobin readily combines with gas or any other substances to form some
products, which are called the derivatives of hemoglobin. The following are the deriva-
tives of hemoglobin.
1. OXYHEMOGLOBIN
This is formed by the combination of hemoglobin with oxygen by the physical
process of oxygenation. Oxyhemoglobin is an unstable compound and the combination
is reversible, i.e. the oxygen can be released from this compound. The iron remains in
ferrous state in this compound.
2. REDUCED HEMOGLOBIN OR FERROHEMOGLOBIN
When oxygen is released from oxyhemoglobin, it is called reduced hemoglobin or
ferrohemoglobin.
3. CARBHEMOGLOBIN
It is the derivative of hemoglobin with carbon dioxide. Carbon dioxide can be
released easily from this. The affinity of hemoglobin for carbon dioxide is 20 times
more than for oxygen.
4. CARBOXYHEMOGLOBIN
The combination of hemoglobin with carbon monoxide produces this. The affinity
of hemoglobin for carbon monoxide is 210 times more than its affinity for oxygen.
Carbon monoxide is a gas produced by the incomplete combustion of hydrocarbons
such as gasoline. It binds to the iron in Hb about 210 times as readily as does oxygen
and does not tend to dissociate. As a result, the Hb bound to carbon monoxide no longer
transports oxygen. Nausea, headache, unconsciousness, and death are possible
consequences of prolonged exposure to carbon monoxide. Cigarette smoke produces
carbon monoxide. If a nonsmoker smoked a pack of cigarettes a day for a few weeks
than carbon monoxide binds to the iron of Hb and prevents the transport of oxygen.
The decreased oxygen stimulates the release of erythropoietin, which increases
erythrocytes production in red bone marrow, causing the number of erythrocytes in the
blood to increase.
5. SULFHEMOGLOBIN
It is formed by the combination of hemoglobin with hydrogen sulfide.
6. NITROUS OXIDE HEMOGLOBIN
It is produced when hemoglobin combines with nitrous oxide.
7. METHEMOGLOBIN OR FERRIHEMOGLOBIN

33
 It is formed when blood is treated with potassium ferricyanide. It is a stable compound.
The iron is in ferric form id est its covalence is III. That is why oxygen binding to Fe is
disturbed and person has hypoxy state. Immature babies, the adult at intoxications (in
part, with methylenic blue, nitrates, nitrites) can have methemoglobin.
STRUCTURE OF HEMOGLOBIN
Hemoglobin is a conjugated protein. It consists of a protein combined with an iron
containing pigment. The protein part is globin and the iron containing pigment is heme.
IRON
It is present in ferrous (Fe++) form. It is in unstable or loose form. Under certain
conditions, the iron may be present in ferric (Fe+++) state, which is a stable form.
GLOBIN
This contains four polypeptide chains. Among the four polypeptide chains, two are
alpha chains and two are beta chains.
TYPES OF HEMOGLOBIN
Hemoglobin is of two types namely:
1. Adult hemoglobin-HbA
2. Fetal hemoglobin-HbF
There are some structural differences between these two types of hemoglobin. In
adult hemoglobin, the globin contains two alpha chains and two beta chains. In fetal
hemoglobin, there are two alpha chains and two gamma chains instead of beta chains.
ABNORMAL HEMOGLOBIN
Abnormal hemoglobin is produced because of genetic mutation, which leads to
structural variation in the polypeptide chains. There are two categories of abnormal
hemoglobin namely, hemoglobinopathies and hemoglobin in thalassemia and related
disorders.
In hemoglobinopathy, there is structural abnormal in the polypeptide chains. Some of
the hemoglobinopaties are :
1. Hemoglobin S: This is found in sickle cell anemia. The alpha chains are normal and beta
chains are abnormal.
2. Hemoglobin C: This occurs in hemoglobin C disease. Here, the beta chains are
abnormal.
3. Hemoglobin E: This occurs in hemoglobin E disease. Here also the beta chains are
abnormal.
In thalassemia, the polypeptide chains are decreased absent or abnormal. In alpha-
thalassemia, the alpha chains are decreased, absent or abnormal and in beta- thalassemia
the beta chains are decreased, absent or abnormal.
DESTRUCTION OF HEMOGLOBIN
After the lifespan of 120 days, the red blood cell is destroyed in the
reticuloendothelial system particularly in spleen and the hemoglobin is released into
plasma. Soon, the hemoglobin is degraded in the reticuloendothelial cells and split into
globin, iron and porphyrin.
IRON
Iron is stored in the body as ferritin and hemosiderin, which are reutilized for
synthesize of new hemoglobin.
GLOBIN
34
 It is utilized for the resynthesis of hemoglobin.
PORPHYRIN –(Hb pigment part consisting of 4 pyrole rings)
It is converted into a green pigment called biliverdin. In human being, most of the
biliverdin is converted into a yellow pigment called bilirubin. Bilirubin and biliverdin
are together called the bile pigments.
IRON METABOLISM
IMPORTANCE OF IRON
Iron is important for the formation of hemoglobin, myoglobin and other substances
like cytochrome, cytochrome oxidase, peroxidase and catalase.
NORMAL VALUE AND DISTRIBUTION OF IRON IN THE BODY
The total quantity of iron in the body is about 4 grams. The approximate distribution
of iron in the body is as follows:
65 to 68% : In the hemoglobin.
4% : In the muscle as myoglobin.
1% : In the form of various heme compounds, which take part in the intracellular
oxidation.
0.1% : In the plasma as transferrin.
25to 30% : Stored in the reticuloendothelial system and liver in the form of ferritin.
DIETARY IRON
Dietary iron is available in two forms called heme and nonheme. Heme iron is present
in fish, meat and chicken.
Nonheme iron is available in vegetables, grains, and cereals.
ABSORPTION OF IRON
Iron is mainly absorbed from the small intestine. Bile is essential for the absorption
of iron. Iron is absorbed through the intestinal cells by pinocytosis and transported into
the plasma.
TRANSPORT AND STORAGE OF IRON
Immediately, after absorption into the blood, iron combines with a beta globulin called
apotransferrin to form transferrin and is transported in this form in the plasma. Iron
combines loosely with the globin and can be released easily at any region of the body.
Iron is stored in large quantities in the reticuloendothelial cells and liver hepatocytes.
In other cells also, it is stored in small quantities. In the cytoplasm of the cell, iron
combines with a protein and forms apoferritin, which is converted into ferritin and is
stored in this form in large amount. Small quantity of iron is also stored as hemosiderin,
which is highly insoluble.
DAILY LOSS OF IRON
In males, about 1 mg of iron is excreted everyday through feces. In females, the
amount of iron lost from the body is very much high. This is because of the
menstruation.
1 g of hemoglobin contains 3.34 mg of iron. Normally, 100 ml of blood contains 15 g
of hemoglobin and about 50 mg of iron (3.34 x 15). So, if 100 ml of blood is lost from
the body, there is a loss of about 50 mg of iron. In females, during every menstrual
cycle, about 50 ml of blood is lost by which 25 mg of iron is lost. That is why the iron
content of blood is always less in females than in males.

35
 Iron is lost during hemorrhage also. If 450 ml of blood is donated, about 225 mg of
iron is lost.
REGULATION OF TOTAL BODY IRON
Absorption and excretion of iron are maintained almost equally under normal
physiological conditions. When the iron storage is saturated in the body, it
automatically reduces the further absorption of iron from the gastrointestinal tract by
feedback mechanism. The factors, which reduce absorption of iron, are:
1. Stoppage of apotransferrin formation in the liver, so that, the iron could not be
absorbed from the intestine and
2. Reduction in the release of iron from the transferrin so that, transferrin is completely
saturated with iron and further absorption is prevented. This type of regulation is
known as feedback mechanism.

HEMOLYSIS AND FRAGILITY OF RED BLOOD CELLS

HEMOLYSIS : 1) the destruction of formed elements;
2) the process which involves the breakdown of red blood cell and liberation of
hemoglobin.
FRAGILITY – the susceptibility (to be affected) of RBC to hemolysis or tendency
to easy breakability of RBC (fragile=easily broken). 2 types:
a) osmotic – occurs due to exposure to hypotonic saline;
b) mechanic – due to mechanic trauma (during wound or injury).

HEMOLYSIS PROCESS
Plasma and RBC are in osmotic equilibrium. The fluids inside and outside the cell
are separated by cell membrane. When the osmotic equilibrium is disturbed, the cells
are affected. For example, when the RBC are immersed in hypotonic solution there is
endosmosis, id est water enters the cell. The cells swell up and are damaged by
bursting. And the Hb is released from the ruptured Er.

HEMOLYSIS TYPES:
1) intracellular – due to macrophages action;
2) intravascular:
• osmotic – in hypotonic solution (minimal when the least stable RBC are
destructed – at 0,42-0,48% NaCl, maximal or complete when the most
stable cells are destroyed –at 0,30-0,34% NaCl) – at anemias minimal and
maximal boarders are replaced to the side of hypotonic saline
concentration rising;
• chemical;
• biological – at snakes venom action;
• mechanic – at ampule (with blood) shaking; in patients with heart and
vessels valves; at durable walking because of Er traumatizing in feet
capillaries (Hb and its derivatives appeared in urine leading to marsh
hemoglobinury);
36
 • thermal – if one frozens Er and then heals them;
• immune – at incompatible blood transfusion and at autoantibodies
presence.

CONDITIONS WITH HEMOLYSIS

- hemolytic jaundice;
- antigen-antibody reactions;
- poisoning by chemicals or toxins;
- while using artificial kidney for hemodialysis;
- while using heart-lung machine during cardiac surgery.

HEMOLYSINS OR HEMOLYTIC AGENTS – the substances which cause

destruction of erythrocytes. There are 2 types:
1) chemical substances:
a) alcohol, benzene, chloroform and ether;
b) acids (acetic et al.), alkalis (like ammonia), bile salts and saponin;
c) chemical poisons like arsenial preparations, carbonic acid, nitrobenzene and
resin;
2) substances of bacterial origin or substances found in body:
a) toxic substances or toxins from bacteria like Streptococcus, Staphylococcus,
Bacillus tetani etc.;
b) venom of poisonous snakes like cobra.

Materials for auditory self-work.

4.1. List of study practical tasks necessary to perform at the practical class.
It’s necessary for work: microscope, counter of Goryaev, scarificators, mixers,
solutions, alcohol, cotton wool, photoelectrocalorymeter (PEC), apparatus for
erythrocytes automatic count, hemometer of Sali, hydrochloric acid solution, alkaline
solution, device for hemolysis assessment.

Task 1. To determine erythrocytes amount in blood.

In some clinics, unfortunately, this old method is still widely-used. Under
modern conditions this method was changed on automatical one. Automatical devices
have special instructions on their expluatation.
Erythrocytes number estimation in Goryaev’s chamber.
To petch investigated blood in special mixer (melanger) till the mark 0,5 or 1,0 (it
depends on blood dissolving). To wipe mixer end with cotton wool and to petch 3%
NaCl in it till the mark “101”. To mix carefully mixer content in course of 1 minute, to
pour 1-2 blood drops on cotton wool and to fill up Goryaev’s chamber with the next
blood drop. But one should grind covering glass to Goryaev’s chamber before this.
Erythrocytes are estimated in 5 large squares (each of them is divided into 16 small
squares) placed diagonally (obliquelly) to the net. It’s necessary to estimate red blood
cells located inside every small square as well as on its superior and left boundaries.
One should put found erythrocytes amount under following formula:
37
 X= (a x 4000 x 200 or 100) : 80 x 106,
where:

X – erythrocytes amount,
a - erythrocytes amount in 5 large (80 small) squares.
1/4000 mcl/mm3 – one small square volume;
200 or 100 – blood dilution degree;
106 – co-efficient for re-computation into SI.

Task 2. Hemoglobin content determining in blood.

Like in a case with erythrocytic amount, there are both rutine (old) measurements
methods and new, automatical ones (on PEC, hemoglobinometers and others).
Hemoglobin content determination by Sali method.
To pour 0,1 normal (approximately 0,2 ml) hydrochloric acid solution in graduated
pipette of Sali till inferior ring level. To petch exactly 0,02 ml of blood with pipette
from hemometer and to blow it on the test tube floor. Shaking up the test tube, to mix
its content carefully. The mixture must stay in course of 5-10 minutes at room
temperature. Hydrochloric acid causes erythrocytic hemolysis and hemoglobin
destruction. Releasing hem interacts with hydrochloric acid and is transformed into
hydrochloric hematin. Test tube content becomes dark brown as a result of this
reaction. The investigator must add distillate water in 5-10 min till investigated liquid
color becomes equal to the standard solution color. To mark on the scale at which level
hydrochloric hemolytic is. Received ziphra multiple on 10. Result (product) will
correspond to hemoglobin concentration in investigated blood in g/l.

Task 3. To estimate blood color index.

Color index characterizes erythrocytes satiation degree with hemoglobin. It is
calculated on formula:
C.I.= (X hemoglob. x 5,0 x 1012/l) : (167 g/l x X erythroc.), where:
X hemoglob. – found hemoglobin amount (g/l);
X erythroc.- found erythrocytes amount in 1 l of blood.
The second formula: Hb (g/l) x 3: RBC (3 first ziphras). It is evaluated in conditional
units.
Normal values:
• 0,75-1,0 – erythrocytic normochromy;
• more than 1,0 – hyperchromy;
• less than 0,75 – hypochromy.

Literature recommended:
1. Lecture course.
2. Mistchenko V.P., Tkachenko E.V. Methodical instructions for medical students
(short lecture course).-Poltava, 2005.-P. 62-65.

38
 3. Mistchenko V.P., Tkachenko E.V. Blood system Physiology //Methodical
recommendations to practical classes for students of medical and dental
departments.-Poltava, 2005.-20p.
4. Kapit W., Macey R.I., Meisami E. The Physiology Colouring Book: Harpers
Collins Publishers, 1987.-P. 136.
5. Guyton – Ganong – Chatterjee. Concise Physiology /Ed. By Dr Raja Shahzad
Gull: M.B.B.S., F.C.P.S., King Edward Medical College.-Lahore, 1998 (1st
Edition).-P.171-178, 204.
6. Stuart Ira Fox. Human Physiology.-8th Ed.-McGrawHill, 2004.-P.368-369, 371-
372.
7. Seeley R.R., Stephens T.D., Tate P. Essentials of Anatomy and Physiology.-The
3rd Ed.-McGraw Hill, 1999.-P.288-291, 299.

Materials for self-control:

Control questions:
1. Erythrocytes structure and quantity, their amount changings under physiological
conditions.
2. Erythrocytes functions.
3. Erythropoiesis regulation, specific and non-specific erythropoiesis regulative
ways. Salivary glands role in this process.
4. Hemoglobin molecule structure, hemoglobins types.
5. Hemoglobin functions.
6. Hemoglobin chemicals in blood.
7. Color index.
8. Erythrocytic hemolysis, its types.
9. Hemolysins.
10. Different environments and solutions influence on erythrocytic hemolysis.
LESSON 33
BLOOD GROUPS BELONGING INVESTIGATION

1. The topic studied actuality. Data about blood grouping is essential in clinical
and theoretical medicine as well as in people' everyday life. Blood groups can be
determined in saliva because there are proteins similar structurally to the ones of
erythrocytes agglutinogens of human being corresponding blood group.
Blood groups role in medicine
I. Identic blood groups are only in one-egged twins. Anjes has proposed blood-
replacers in 1838. Blood transfusion leads to hepatitis A, B, C (25%), AIDS. Cancer
rate is increased after blood transfusions (recidives, metastases et al.) as well as the one
of many infectious diseases. Artificial blood does not have antigenic features. If blood
loss is from 800 ml to 1500 ml than measurements must not be done. Blood comes
from depot. Deponated blood is viscous because it contains many erythrocytes. That is
why doctor must inject 0,9% of NaCl and the patient should drink much.
Autotransfusions are rather useful during operations: the patient gives his blood that is

39
given him back during the surgery. Blood transfusions are possible only at acute
surgical situations and at obstetric-gynecological ones.
There are 3 rules of hemotransfusion:
1) the donor environment must correspond to the one of recipient: one must
describe agglutinogens content in donor (they are very stable) and agglutinins in
recipients;
2) donor and recipient blood must be compatible.
Universal donor (one-grouped blood) and recipient must not be because there are
more than 500 antigens on erythrocytic membrane (400 mln of combinations). If to add
other blood antigens than there will be 700 bln of combinations. Thus, blood is different
in every person (because combinations amount is more than people on Earth).
Agglutination must be absent at donor's blood mixture with the recipient's one.
3) Only the freshest, warm blood can be transfused. Plasma is less antigenic.
II. Transplanting problem. Every cell allows assessment blood group. For instance,
if agglutinogen A is present in blood it is present in every cell. Organs banks are
created.
III. Blood groups and diseases:
I (0): - ulcer disease of stomach and duodenum – agglutinogens A and B having
been released in stomach and pancreatic juice content prevent wall from proteolytic
enzymes (these agglutinogens are absent in people with the I-st blood group):
- abscesses;
- lymphadenopathies;
- syphilis;
- liver cirrhosis;
- cholecystitis;
- appendicitis;
- ulcerous stomatitis;
- gingivitis (and other inflammatory disease);
- pernicious anemias;
- acute respiratory viral infections;
- excessive bleedings (blood coagulation weakening);
II (A): - thromboses (excessive blood coagulation);
- diabetes mellitus;
- heart-vascular system diseases in part hypertonic disease;
- myocardial infarction and strokes (due to enforced blood coagulation);
III (B): - infectious (especially hard and atypic, tropical);
IV (AB): - tumors;
- thrombotic states (less than in people with the A (II).
Rh-negative people have blood diseases more often in 6 times in comparison to Rh-
positive people. Patients with congenital heart vices complicated with infectious
endocarditis are most often the ones with negative rhesus. O(I)Rh- often have
paroxysmal night hemoglobinury (disease of Markiafava-Mikelli), innate hemolytic,
hypoplastic and aplastic anemias.
People with II (A), IV (AB) are drug-dependent more than the others.

40
 Although one must take into account that links between blood groups and morbidity
carry not direct character but the one mediated by other factors. More visible
dependence is present between leucocytes antigens (HLA) and especially the ones of
the II-nd class and predisposition to different diseases. This fact is determined by the
following: HLA defines immune response intensiveness as well as answer for local
immunity in separate organs.
Rh-conflict can lead to miscarriages, fetus intrauteral death, immaturity, toxicoses
of mothers.
Paternity canceling – on the base of MNS-system genes.
Forensic medicine – hairs, urine, sperm or other organs and tissues contains
antigens that can prove or disprove personality.
Blood groups significance in theoretical medicine and everyday life
Anthropology deals with blood grouping because every race, even simple ethnic
groups can be differentiated by one or another blood group dominance. For example,
seldom antigen Diego has been discovered in Polynesia inhabitants. Syria Arabs
possess O(I) and A(II) more often than B(III). IV(AB) is practically absent in them.
90% of American Indians have 0(I).
2. Study aims:
To know: main blood group by ABO-system and Rh-belonging, all probes
performance principles (probes before blood transfusion).
To be able to: determine blood group by ABO system with standard sera, Tsoliclones
anti-A and anti-B as well as Rh-belonging with Tsoliclones anti-C, anti-D and anti-E.

3. Pre-auditory self-work materials.

3.1.Basic knowledge, skills, experiences, necessary for study the topic:
Subject To know To be able to
Biophysics Data about erythrocytes Tell about RBC agglutination
agglutination and hemolysis mechanism
Medical Biology Data about blood groups Solve tasks on blood groups
inheritance inheritance
Hemolytic disease Tell about newborns hemolytic
Pathophysiology developmental mechanisms disease developmental
mechanisms, ABO and Rh-
conflict ways and results
Pediatry with Blood groups inheritance Tell about newborns hemolytic
Neonatology mechanisms disease developmental
mechanisms, ABO and Rh-
conflict ways and results
Neurology with Data about blood groups Solve tasks on blood groups
Medical Genetics inheritance inheritance
Obstetrics and Data about blood groups Prevent and treat ABO and Rh-
Gynecology inheritance, newborns hemolytic conflict as well as newborns
disease developmental hemolytic disease
mechanisms, ABO and Rh-
conflict ways and results,
41
 hemotransfusiology biological
bases
Surgery Hemotransfusiology biologicalTo perform hemotransfusions,
bases blood preparations and
components transfusions as well
Anesthesiology Data about blood groups To perform hemotransfusions,
and inheritance, hemotransfusiology blood preparations and
Rheanimatology biological bases components transfusions as well

3.2. Topic content

Historical data
1666 – London anatomist and physiologist Richard Lower transfused blood from
one dog to another one and proved that hemotransfusions are possible to be applied for
replacing therapy.
1666 - sheep blood has been transfused to the human being with significant
improvement in France. The results were good may be because of transfused blood
little amount (French doctor Zhan Deni).
1667 – two English doctors King and Kox transfused blood from animals of one
species to the animals of another species and proved that these manipulations are very
dangerous.
But about 20 hemotransfusions have been realized in the XVII-th century from
animals to the human beings. Not all of them were with positive results.
1819 – English obstetrician Blendel has performed the first hemotransfusion from
one human being to another one. He made transfusion to the woman dying from
hemorrhagy after labors. Blood has been taken from her husband. The transfusion
result was successful. Then the doctor has performed 11 hemotransfusions else to the
women under similar conditions.
Obstetrician Wolf did the same in Russia.
There were about 600 hemotransfusions in the XIX-th century.
1848 – the first manuel on hemotransfusions. It was in Russia. Its name was
“Tractate about blood transfusion as the only meaning how to save decreasing life
having been compiled in historical, physiological and surgical aspects”. Its author was
Alexei Philomafitsky.
Blood groups are determined by protein molecules present on the surface of red
blood cells. When blood from two individuals is mixed, sometimes clumping
(agglutination) occurs. This clumping is because of the immunological reactions. But,
why clumping occurs in some cases and not in other cases remained a mystery until the
discovery of blood groups by Karl Landsteiner in 1901. He was honored with Nobel
Prize in 1930 for this discovery. The immunological reaction is the antigen- antibody
reaction. Landsteiner found two antigens or agglutinogens in red blood cells and named
them as A antigen and В antigen. He noticed the corresponding antibodies or
agglutinins in the serum called a antibody or anti A and (3 antibody or anti B. However,
in the body, a particular antigen and the corresponding antibody cannot be present
together. If present, it causes clumping of the blood.

42
 Karl Landsteiner, the American scientist of Austrian origin has told about 3 blood
groups (ABO system) in 1901. But soon he has told about seldom forth blood group.
1907 (practically in the same time) – Czech scientist Yan Yansky has informed about
blood group signs on the background of which one can solve the question about blood
transfusion from one human being to another one. It is important to mention that the
scientist did not know about discovery made by Karl Landsteiner.
1926 – Institute of hemotransfusions has been opened in Moscow.
1940 – K. Landshteiner and his follower A.Winer have found antigen in monkey-
rhesus blood and called it Rhesus-factor.

The ABO blood types are not found in equal numbers. For instance, in whites in the
USA the distribution is type O – 47%, type A – 41%, type B – 9%, and type AB – 3%.
Among blacks in the United States the distribution is type O – 46%, type A – 27%,
type B – 20% and type AB – 7%.

ANTIGENICITY AND IMMUNE REACTIONS OF BLOOD

At least 30 commonly occurring antigens, each of which can at times cause antigen-
antibody reactions, have been found in human blood cells, especially on the surfaces
of cells membranes. In addition to these, more than 300 others of less potency or that
occur in individual families rather than having widespread occurrence are known to
exist. Among the 30 or more common antigens, certain ones are highly antigenic and
regularly cause transfusion reactions if proper precautions are not taken, whereas others
are of importance principally for studying the inheritance of genes and therefore for
establishing parentage, race, and so forth. Essentially all these antigens are either
glycolipids or glycoproteins.
Bloods are divided into different groups and types in accordance with the types of
antigens present in the cells. Two particular groups of antigens are more likely than
others to cause blood transfusion reactions. These are so-called O-A-B system of
antigens and the Rh system.
ABO SYSTEM
Two related antigens – type A and B – occur on the surfaces of the RBC in a large
proportion of the population. Because of the way these antigens are inherited, people
may have neither of them on their cells, they may have one, or they may have both
simultaneously.
Strong antibodies that react specifically with either the type A or type B antigen
almost always occur in the plasmas of persons who do not have the antigens on their
red blood cells. These antibodies bind with the red cell antigens to cause agglutination
of the red cells. Therefore, the type A and type B antigens are called agglutinogens,
and the plasma antibodies that cause the agglutination are called agglutinins.
When neither A nor B agglutinogen is present, the blood group is group O (I). When
only type A agglutinogen is present, the blood is group A(II). When only type B
agglutinogen is present, the blood is group B(III). And when both A and B
agglutinogens are present, the blood is group AB(IV).
When type A agglutinogen is not present in a person's red blood cells, antibodies
known as “anti-A” agglutinins develop in his plasma. Also, when type B agglutinogen
43
is not present in the red blood cells, antibodies known as “anti-B” agglutinins develop
in the plasma.

TABLE 2. The blood groups with their genotypes and their constituent
agglutinogens and agglutinins
Genotypes Blood groups Agglutinogens Agglutinins
00 0 (I) - Anti-A and Anti-
B
0A or AA A (II) A Anti-B
0B or BB B (III) B Anti-A
AB AB(IV) A and B -

The agglutinins are gamma-globulins, as are other antibodies, and are produced by
the same cells that produce antibodies to any other antigens. Most of them are Ig G and
M immunoglobulin molecules.
But why are these agglutinins produced in individuals who do not have the antigenic
substances in their red blood cells? The answer to this seems to be that small amounts
of group A and B antigens enter the body in the food, in bacteria, and in other ways,
and these substances initiate the development of the anti-A or anti-B agglutinins. One
of the reasons for believing this is that injection of group A or group B antigen into a
recipient having another blood type causes a typical immune response with formation
of greater quantities of agglutinins than ever. Also, the newborn baby has few if any
agglutinins showing that agglutinin formation occurs almost entirely after birth.
Agglutinins are designated by letters б- and в-. Blood of one individual can not have
similar agglutinogens and agglutinins. There are also hemolysines in plasma and serum
(they are designated like agglutinins). One can see conflict at blood hemotransfusions
at meeting of one-named agglutinogens and hemolysines (they act at 37-40°C). At
room temperature, if one-named agglutinogens and agglutinins meet each other
agglutination reaction occurs –the criterium of group characteristic. So, antigen-
antibody interaction at room temperature is known as agglutination, at increased one –
hemolysis. Both variants are accompanied by cells clumping.
High resistance to temperature, blood preservation terms are the characteristic of all
agglutinogens. That’s why they contain practrically in all tissues of given organism
and its fluids. That’s why agglutinogens content is essential to be known, when blood
is received from donor with its further usage for transmission. On the contrary,
agglutinins are unstable comparatively to agglutinogens and they are easily destroyed
while contact with side surface, while temperature changing. That’s why they are not
important in donor blood, but their determining is quite essential in recipient blood.
A donor is a person who gives blood, and a recipient is a person who receives blood.
Usually a donor can give blood to a recipient if they both have the same blood type.
For example, a person with type A blood could donate to another person with type A
blood. There would be no ABO transfusion reaction because the recipient has no
antibodies against the type A antigen. On the other hand, if type A blood were donated
to a person with type B blood, a transfusion reaction would occur because the person

44
with type B blood has antibodies against the type A antigen, and agglutination would
result.
Historically, people with type O blood have been called universal donors because
they usually can give blood to the other ABO blood types without causing an ABO
transfusion reaction. Their erythrocytes have no ABO surface antigens and therefore
do not react with the recipient's A or B antibodies. For example, if type O blood is
given to a person with type A blood, the type O erythrocytes do not react with the type
B antibodies in the recipient's blood. In a similar fashion, if type O blood is given to a
person with type B blood, there would be no reaction with the recipient's type A
antibodies.
It should be noted, however, that the term universal donor is misleading. In two
circumstances transfusion of type O blood can produce a transfusion reaction. First,
mismatching blood groups other than the ABO blood group can cause a transfusion
reaction. To reduce the likelihood of a transfusion reaction, all the blood groups must
be correctly matched. Second, antibodies in the blood of the donor can react with
antigens on the erythrocytes in the blood of the donor can react with antigens on the
erythrocytes in the blood of the recipient. For example, type O blood has type A and B
antibodies. If type O blood is transfused into a person with type A blood, the A
antibodies (in the type O blood) react against the A antigens (on the erythrocytes in the
type A blood). Usually such reactions are not serious because the antibodies in the
donor's blood are diluted in the blood of the recipient, and few reactions take place.
Because type O blood causes transfusion reactions in these situations, however, it is
given to a person with another blood type only in life-or-death conditions.
Historically, people with type AB blood were called universal recipients. People
with type AB blood were called universal recipients because they could receive type
A, B, AB, or O blood with little likelihood of a transfusion reaction. Type AB blood
does not have antibodies against type A or B antigens. Transfusion of these antigens in
type A, B, or AB blood does not therefore cause a transfusion reaction in a person with
type AB blood. The term is misleading, however, for two reasons. First, other blood
groups can cause a transfusion reaction. Second, antibodies in the donor's blood can
cause a transfusion reaction. For example, type O blood contains A and B antibodies
that can react against the A and B antigens in type AB blood.

Rh-SYSTEM
Rhesus-system was discovered in the middle of last century. Rh blood group so
named because it was first studied in the rhesus monkey. 85 per cent of people have
agglutinogen of this system (Rh-rhesus) and these people are called rhesus-positive. 15
per cent of people have no this antigen and correspondingly they are known as rhesus-
negative. 88% of black people in the United States are Rh-positive. The ABO blood
type and the Rh blood type usually are designated together. For example, a person
designated as A positive is type A in the ABO blood group and Rh-positive. The rarest
combination in the United States is AB negative, which occurs in less than 1% of all
Americans.
Rh-system is rather complicated, it includes more than 40 antigens. This factor is
inherited. Antibodies against the Rh antigens do not develop unless the Rh-negative
45
person is exposed to Rh-positive erythrocytes. This can occur through a transfusion or
by the transfer of blood across the placenta to a mother from her fetus. When a Rh-
negative person receives a transfusion of Rh-positive blood, the recipient becomes
sensitized to the Rh antigens and produces Rh antibodies. If the Rh-negative person is
unfortunate enough to receive a second transfusion of Rh-positive blood after
becoming sensitized, a transfusion reaction results.
Rh incompatibility can pose a major problem in some pregnancies, when the mother
is Rh-negative and the fetus is Rh-positive. One can say about Rh-conflict (if mother
is Rh-negative, father is Rh-positive than fetus will be Rh-positive as well) and the
newborns hemolytic disease.
1. Before or during delivery. Rh-positive erythrocytes from the fetus enter the blood
of an Rh-negative woman through a tear in the placenta.
2. The mother is sensitized to the Rh antigen and produces Rh antibodies. In the
first pregnancy, there is often no problem. The leakage of fetal blood is usually the
result of a tear through placenta that takes place either late in the pregnancy or during
delivery. Thus there is not enough time for the mother to produce sufficient numbers
of Rh antibodies to harm the fetus.
3. During a subsequent pregnancy with an Rh-positive fetus, Rh-positive
erythrocytes cross the placenta, enter the maternal circulation, and stimulate the mother
to produce antibodies against the Rh antigen. Antibody production is rapid because the
mother has been sensitized to the Rh antigen. The Rh antibodies from the mother cross
the placenta, causing agglutination and hemolysis of fetal erythrocytes, and hemolytic
disease of the newborn (HDN) develops. It can be fatal for the fetus.
Prevention of hemolytic disease of the newborn is often possible if the Rh-negative
woman is given an injection of a specific type of antibody preparation called anti-
Rh0(D) immune globulin Rh0GAM). The injection can be given during the pregnancy,
before delivery, or immediately after each delivery, miscarriage, or abortion. The
injection contains antibodies against Rh antigens. The injected antibodies bind to the
Rh antigens of any fetal erythrocytes that may have entered the mother's blood. This
treatment inactivates the fetal Rh antigens and prevents sensitization of the mother.
If HDN develops, treatment consists of slowly removing the blood of the fetus or
newborn and replacing it with RH-negative blood. The newborn's skin is also exposed
to fluorescent light because it helps to break down bilirubin in the blood as the blood
flows through the skin. The bilirubin is derived from the hemoglobin released from
ruptured erythrocytes. High levels of bilirubin are toxic to the nervous system and can
cause destruction of brain tissue.
Other antigen systems are more seldom (Luteran, Daffi, Kell-Kellano, MNS et al.).
Scientists tell nowadays about 500 antigens only on erythrocytic membrane. If to add
others to them, then their amount will predominate number of all residents on the Earth.
With other worlds, every person has his or her own blood group that is quite essential
to know and to use in clinical practice.
4. Materials for auditory self-work
4.1. List of study practical tasks necessary to perform at the practical class.

46
 Materials and methods: blood, china plate, scarificators, standard group specific
sera, subject glasses, glass sticks, antirhesus serum, Tsoliclones anti-A and anti-B.
Investigative object: human being.

Task 1. To determine human being blood group on ABO system:

A. By means of group-specific sera:
To pour blood sera on china plate correspondingly to blood groups designations. To
process the finger and to prick it with scarificator. To place blood drop in a plate central
nest. To add blood to the serum (in a correlation of 1:10) with clean subject glass
separate angles. To get the plate rocking in course of 3-5 minutes. To mark the nets
where agglutination reaction occurred. To determine blood group.
B. By means of Tsoliclones anti-A and anti-B:
To pour Tsoliclones anti-A and anti-B on 1 big drop (0,1 ml) on the plate under
corresponding writings. To pour investigated blood near drops in 10 times less than
antibodies drop. To mix with glass stick (different in every drop). To get the plate
rocking in course of 2-3 minutes. The result in every drop may be positive or negative.
To determine blood group.
Results interpretation:
• if agglutination reaction is absent with all group-specific sera and with all (both)
Tsoliclones, than given blood group doesn’t contain antigens A and B, thus it
belongs to blood group 0(I);
• if agglutination reaction took place with I and III serum and Tsoliclone anti-A, then
given group contains antigen A and belongs to A(II) group;
• if agglutination reaction occurred with I and II sera and with Tsoliclone anti-B, then
given blood group belongs to B(III) blood group;
• if agglutination reaction took place with sera of I, II, III groups and with both
Tsoliclones, then investigated blood contains both antigens A and B and blood
belongs to the group AB (IV).

Task 2. To determine rhesus-factor while express-method usage.

To pour 1 drop (20 divisions of Panchenkov’s capillary pipette) of anti-rhesus serum
to the investigated blood on test tube floor. To shake up the test tube and to turn over
several times so that its content was flowing on the walls. To add 2-3 ml of 0,85%
solution of NaCl solution in 3 min. To mix test tube content after its 1-2-folded turning
over. Don’t shake up!
To perform results assessment on agglutination absence or presence (large flakes on
the background of enlighten liquid).

Task 3. To perform probe on individual compatibility.

To pour recipient blood plasma on subject glass. To add donor blood drop less in 10
times than plasma (in a correlation of 1:10) to this plasma. To evaluate their
compatibility.

5. Literature recommended:
47
 1. Lecture course.
2. Mistchenko V.P., Tkachenko E.V. Methodical instructions for medical students
(short lecture course).-Poltava, 2005.-P. 70.
3. Mistchenko V.P., Tkachenko E.V. Blood system Physiology //Methodical
recommendations to practical classes for students of medical and dental
departments.-Poltava, 2005.-20p.
4. Kapit W., Macey R.I., Meisami E. The Physiology Colouring Book: Harpers
Collins Publishers, 1987.-P. 137.
5. Guyton – Ganong – Chatterjee. Concise Physiology /Ed. By Dr Raja Shahzad
Gull: M.B.B.S., F.C.P.S., King Edward Medical College.-Lahore, 1998 (1st
Edition).-P.192-196, 208-209.
6. Stuart Ira Fox. Human Physiology.-8th Ed.-McGrawHill, 2004.-P.372-374.
7. Seeley R.R., Stephens T.D., Tate P. Essentials of Anatomy and Physiology.-The
3rd Ed.-McGraw Hill, 1999.-P.296-299.

6. Materials for self-control:

Control questions.
1. Blood groups discovery.
2. Representations about erythrocytic antigens.
3. Data about blood groups systems.
4. Agglutinogens and agglutinines. Main principles of blood division on groups.
5. Blood transfusion rules.
6. Rhesus-factor and its importance for clinics.
7. Knowledge about blood groups significance for doctors of different specialities and
for any human being in their daily life.

LESSON 34
LEUCOCYTES NUMBER, LEUCOCYTIC FORMULE INVESTIGATION

1. The topic studied actuality. Oral cavity performs multiplied specific and non-
specific defence reactions. Neutrophilopeny and enforced phagocytosis is observed at
purulent inflammation in part caused by streptococci and staphylococci, more seldom
– pseudomonas. These microbes are always present in oral cavity. Inflammatory
diseases in maxillar-facial area are rather spread. At some acrylic prostheses (dentures)
one can see eosinophyly. Periodontites can be accompanied by monocytopeny. Viral
infections (in part, herpetic) are connected with interpheron synthesis reducing.
Phagocytes and complement participates in protective reactions at pulpitis,
periodontitis and other dental diseases.

2. Study aims:

48
 To know: separate leucocytes structure, normal value and number, leucopoiesis and
its regulation, organism specific and non-specific reactions.
To be able to: to assess white blood state, leucocytic formule at organism different
functional states.
3. Pre-auditory self-work materials.

3.1.Basic knowledge, skills, experiences, necessary for study the topic:

Subject To know To be able to
Biology and Inheritance main ways Analyze primary
Medical Genetics immunodeficiencies
inheritance ways
Pathophysiology Leucopenies, leucocytoses, Interpret leucocytic formule
immunodeficiencies reasons to say in part about leucocytic
and developmental mechanisms gap, acute and chronic
leucoses
Pediatry with Leucocytes, specific and Say about leucocytes,
Neonatology unspecific defense mechanisms organism defensive
peculiarities in children of mechanisms pathology
different age
Internal Diseases Different leucocytic sets Tell about ethiology,
functions, leucocyte formule, pathogenesis, treatment and
specifc and non-specific prophylaxy of leucopenies,
defencive mechanisms leucocytoses,
immunodeficiencies
Immunology Different leucocytic sets Tell about ethiology,
functions, leucocyte formule, pathogenesis, treatment and
specific and non-specific prophylaxy main primary and
defensive mechanisms, secondary
immunogram major indexes immunodeficiencies

3.2. Topic content

White blood cell (WBC) or leukocyte is the colourless and nucleated formed
element of blood. Leukocytes play very important role in defense mechanism of the
body. Depending upon the presence or absence of granules in the cytoplasm, the
leukocytes are classified into 2 types namely:
1. Granulocytes—with granules and
2. Agranulocytes—without granules.
The granulocytes are neutrophils, eosinophils and basophils. Agranulocytes are
monocytes and lymphocytes (Fig. 6).
MORPHOLOGY OF WHITE BLOOD CELLS
NEUTROPHILS
Neutrophils or polymorphs have fine or small granules in the cytoplasm. The granules
take acidic and basic stains. So, the granules appear violet in colour after staining. The
nucleus is multi-lobed. The number of lobes in the nucleus depends upon the age of
cell. In younger cells, the nucleus is not lobed. And in older neutrophils, the nucleus
49
has 4 or 5 lobes. The diameter of cell is 10 to 12 microns. The neutrophils are ameboid
in character.

EOSINOPHILS
Eosinophils have coarse (larger) granules in the cytoplasm, which stain bright red,
or orange with eosin. The nucleus is bi-lobed. The diameter of the cell varies between
10 and 14 microns.
BASOPHILS
Basophils also have coarse granules in the cytoplasm. The granules stain purple blue
with basic dyes like methylene blue. Nucleus is bi-lobed. Diameter of the cell is 8 to 10
microns.
MONOCYTES
Monocytes are the largest leukocytes with diameter of 14 to 18 microns. The
cytoplasm is clear without granules. The nucleus is round, oval, horseshoe or kidney
shaped. The nucleus is either in the center of the cell or it is pushed to one side and a large
amount of cytoplasm is seen.
LYMPHOCYTES
The lymphocytes also have clear cytoplasm without granules. The nucleus is oval
or kidney shaped occupying the whole of the cytoplasm.
Depending upon the size, the lymphocytes are divided into two groups as:
1) Large lymphocytes—the younger cells with a diameter of 10 to 12 microns.
2) Small lymphocytes—the older cells with a diameter of 7 to 10 microns.
Depending upon the function, the lymphocytes are divided into 2 types as:
T lymphocytes—concerned with cellular immunity. B lymphocytes—concerned with
humoral immunity.

50
 FIGURE 6: Different white blood cells
NORMAL COUNT OF WHITE BLOOD CELLS
1. Total WBC count (TC): 4,000 to 11,000 /cu. mm of blood
2. Differential WBC count (DC): Given in the Table 3.

Table 3. Normal values of different white blood cells

Leukocyte Percentage Absolute value per cu. mm
Neutrophils 50 to 70 3000 to 6000
Eosinophils 2 to 4 150 to 450
Basophils 0 to1 0 to 100
Monocytes 2 to 6 200 to 600
Lymphocytes 20 to 30 1500 to 2700

VARIATIONS IN THE COUNT OF WHITE BLOOD CELLS

Increase in leukocyte count is known as leukocytosis. Decrease in leukocyte count is
known as leukopenia. The term leukopenia is generally used for pathological
conditions only.
PHYSIOLOGICAL VARIATIONS – physiological leucocytosis

51
 1. Age: In infants, the white blood cell count is about 20,000 per cu mm and in
children, it is about 10,000 to 15,000 per cu mm of blood.
2. Sex: The white blood cell count is slightly more in males. In females, the
leukocytes count is increased during menstruation, pregnancy and parturition.
3. Diurnal variation: The cell count is minimum in early morning and maximum in
the evening
4. Exercise: The white blood cell count is increased slightly during exercise.
5. Sleep: During sleep, the white blood cell count is minimal.
6. Emotional conditions: During emotional conditions like anxiety, the count is
increased.
7. Pregnancy: During pregnancy, the leukocyte count is increased.
8. Food taking: especially after proteinic food taking that is explained by its antigenic
character.
9. Ovulation: insignificant leucocytosis (neutrophyly) at simultaneous eosinophils
amount lowering.
10. Fits: up to 20000 and more independently on reasons.
11. Sharp changing of environmental temperature
PATHOLOGICAL VARIATIONS
All types of leukocytes do not share equally in the increase or decrease in the total
leukocyte count. In general, the neutrophils and lymphocytes vary in opposite
directions.
Leukopenia
The decrease in the total white blood cell count occurs in the following pathological
conditions:
1. Anaphylactic shock
2. Cirrhosis of liver, viral hepatitis in acute phase
3. Disorders of spleen
4. Pernicious anemia
5. Typhoid and paratyphoid and
6. Viral infections: measles, rubeole, influenza
7. Chemicals action (benzol)
8. After irradiation
9. Hypoplastic and aplastic processes
10.Medicines (amidopyrinum, butadionum, rheopyrinum, sulphanilamides,
cytostatics)
10.Endocrine diseases (acromegaly, thyroid pathology)
11.Leucoses (cytostatics overdosage)
12.New-formations metastazing in bone marrow

Lymphocytopeny
1. Primary immune pathology (different-typed agammaglobulinemy, tymome)
2. Blood system pathology (aplastic anemias, leucosis)
3. Kushing's syndrome
4. Kidney insufficiency
5. AIDS – specific symptom
52
 6. Irradiation
7. Corticosteroid therapy and alkylic drugs taking
8. Hard edemas
9. Systemic red lupus
10. Purulent inflammation
11. Tuberculosis

Neutropeny
1. Viral infections
2. Chronic infections
3. After cytostatics taking
4. Irradiation
5. Aplastic and vitamin B12-deficient anemias
6. Agranulocytosis

Leukocytosis
Leukocytosis occurs in the common pathological conditions like:
1. Infections (pneumonia, sepsis, meningitis, erysipeloid): leucocytosis absence in
infectious process acute phase is considered to be unfavorable diagnostic criterium
especially at combination with so-called leucocytic formule shift to the left – see below
2. Allergy
3. Common cold
4. Tuberculosis and
5. Glandular fever
6. Nausea and vomiting – with primarily neutrophils amount increasing
7. Inflammatory processes in part at their purulent character
8. Different organs (myocardium, lungs, spleen, kidney) infarction
9. Vast burnings
10.Bleedings
11.Malignant diseases
12.Blood system diseases (leucosis, polycytemy, lymphogranulomatosis)
13.Infectional mononucleosis and lymphocytosis
14.Uremia
15.Diabetic coma
16.After splenectomy (expressed leucocytosis 15…20 x 109/l with neutrophyly up to
90%)
However, different leukocyte count is increased in specific diseases as given below.

Neutrophilia
The increase in neutrophil count is called neutrophilic leukocytosis. This occurs in
the following conditions:
1. Metabolic disorders
2. Injection of foreign proteins
3. Injection of (foreign) vaccines
4. Poisoning by chemicals and drugs like lead, mercury, camphor, benzene
53
 derivatives, etc.
5. Poisoning by insect venom and
6. After acute hemorrhage.

Eosinophilia
The increase in eosinophil count is called eosinophilia and this occurs in:
1. Allergic conditions
2. Asthma
3. Blood parasitism (malaria, filariasis) and ascaridosis
4. Intestinal parasitism and
5. Scarlet fever
6. Tumors
7. Lymphogranulomatosis
8. Chronic myeloleucosis
9. Medicines (antibiotic, sulphanilamides in part)

Basophilia
Increase in basophil count is called basophilia and it occurs in:
1. Smallpox
2. Chicken pox and
3. Polycythemia vera
4. Allergic processes accompanied by rash
5. Before and during menstrual bleeding
6. Stress
7. Leucosis
8. Myocardial infarction

Monocytosis
Increase in monocytes count is known as monocytosis and occurs in:
1. Chronic infections (tuberculosis, syphilis, brucellosis)
2. Acute infections (rubeole, scarlet fever, infectious parotitis, mononucleosis)
3. Lymphogranulomatosis
4. Endocardites
5. Helminthes
Limphocytosis
1. Increase in lymphocyte count is called lymphocytosis and this occurs in:
2. Diphtheria
3. Infectious hepatitis
4. Mumps
5. Malnutrition
6. Rickets
7. Syphilis
8. Thyrotoxicosis and
9. Tuberculosis

54
 Leukemia
The leukemia is the condition, which is characterized by abnormal uncontrolled
increase in leukocyte count up to 1,000,000/cu mm.

LIFESPAN OF WHITE BLOOD CELLS

Lifespan of white blood cells is not constant. It depends upon the demand in the body
and their function. Lifespan of these cells may be as short as half a day or it may be as
long as 3-6 months.
However, the normal lifespan of white blood cells is as follows:

Neutrophils — 2-5 days

Eosinophils — 7-12 days
Basophils — 12-15 days
Monocytes — 2-5 days
Lymphocytes — 1 day
Table 4. White Blood Cells Lifespan

PROPERTIES OF WHITE BLOOD CELLS

1. Diapedesis
Diapedesis is the process by which the leukocytes squeeze through the narrow blood
vessels.
2. Ameboid Movement
Neutrophils, monocytes and lymphocytes show amebic movement by protruding into
the cytoplasm and changing the shape.
3. Chemotaxis
A number of chemical substances in the tissues cause the leukocytes to move
towards tissues. This phenomenon is called chemotaxis.
4. Phagocytosis
Neutrophils and monocytes swallow foreign bodies by means of pseudopodia.

FUNCTIONS OF WHITE BLOOD CELLS

NEUTROPHILS - are produced in bone-marrow, live 8-10 hours, part of them are
in circulation, another one – into marginal state and significant part leaves blood and
dies in tissues.
Neutrophils play an important role in the defense of the body. Along with monocytes,
the neutrophils provide the first line of defense against the invading microorganisms.
The granules of neutrophils contain enzymes like proteases, myeloperoxidases,
elastases and metalloproteinases. The granules also contain antibody like substances
called defensins. Defensins are antimicrobial peptides, which are active against bacteria
and fungi. Neutrophils also secrete platelet activating factor (PAF) which accelerate the
aggregation of platelets during injury to the blood vessel.
Mechanism of Action of Neutrophils
Neutrophils are released in large number from the blood at the time of infection by the
foreign microorganisms. At the same time, new neutrophils are produced from the

55
progenitor cells. All the neutrophils move by diapedesis and are attracted towards the
site of infection by means of chemotaxis. The chemotaxis occurs due to the attraction
by some chemical substances called chemo-attractants, which are released, from the
infected area. After reaching the area, the neutrophils surround the area and get adhered
to the infected tissues. The chemo-attractants increase the adhesive nature of
neutrophils so that all the neutrophils become stickier and get attached firmly to the
infected area. Now, the neutrophils start destroying the invaders. First, these cells engulf
the bacteria and then destroy them by means of phagocytosis. Each neutrophil can hold
about 15-20 microorganisms at a time. During the battle against the bacteria, many
leukocytes are also killed by the toxins released from the bacteria. The dead cells are
collected in the center of infected area. The dead cells together with plasma leaked from
the blood vessel, liquefied tissue cells and red blood cells escaped from damaged blood
vessel (capillaries) constitute the pus. The dead white blood cells are called the pus cells.
Functions:
• participating in phagocytosis;
• apoptosis triggering;
• interleukines-1,6,8 and 12 formation;
• interpheron formation;
• immune reactions;
• participation in mitosis;
• reparational and regenerational processes;
• hematopoietic reactions;
• blood coagulation;
• fibrinolysis (they contain plasminogen activator).

EOSINOPHILS - are produced in bone-marrow, live from 4 to 12 days. They are

only several hours in blood stream, then penetrate into the tissue for destruction.
Functions:
• phagocytosis;
• antitoxic function;
• kallikrein-kinin system components activation.
Eosinophils are specifically meant for acting against the parasites. The eosinophil
count increases during parasitic infestations and allergic conditions. Rose sundown of
speedy recovery after infectious pathology - eosinophyly – is a very favorable and
long-awaited diagnostic criterium. Eosinopeny – their amount decreasing - is observed
at hard infectional diseases – unfavourable diagnostic sign.
Mechanism of Action of Eosinophils
The eosinophils are neither markedly motile nor phagocytic like the neutrophils. But their
granules contain mar, substances, which become cytotoxic when released during the invading
organisms. Following are the lethal substances present in the granules of eosinophils and
release; at the time of exposure to parasites or foreign proteins
1. Eosinophil peroxidase: This enzyme is capable destroying helminthes (worms),
bacteria and turned cells.

56
 2. Major basic protein (MBP): This is very active against helminthes. It can damage the
parasites by distension (ballooning) and detachment of the tegmental sheath (skin like
covering) of these organism
3. Eosinophil cationic protein (ECP): This substances the major destroyer of helminthes.
It destroys the parasite by means of complete disintegration. It is also acts as
neurotoxin.
4. Eosinophil derived neurotoxin: it destroys the nerve fibers particularly the myelinated
nerve fibers.

BASOPHILS - are formed in bone-marrow, live up to 12 hours. Their relatives - fat

cells (mast cells, mastocytes) live for years.
The basophils play an important role in healing process after inflammation and in acute
hypersensitivity reactions (allergy). The number of basophils is increased during healing
process and right before menstruation (especially in sexual organs vessels).
Mechanism of Action of Basophils
The functions of basophils are executed by the releasing of some important substances
from their granules.
1. Histamine: It produces the acute hypersensitivity reactions by causing vascular
changes and by increasing the capillary permeability.
2. Heparin: Heparin is essential to prevent the intravascular blood clotting.
3. Hyaluronic acid: This is necessary for deposition of ground substances in the
basement membrane. It participates in membrane permeability increasing.
4. Proteases and myeloperoxidase: These enzymes may exaggerate the inflammation
responses.
5. Platelets activation factor synthesis.
6. Thromboxanes production – see next lection.
7. Leucotryens – participate in multiple organism reactions.
8. Prostaglandines – the same + next lecture.

The basophils also have IgE receptors, which het; them to produce hypersensitivity
responses.
It is necessary to mention that eosinophils can reduce allergy reactions because they
contain histaminase – histamine-decomposing enzyme. There is so-called eosinophylic-
basophylic association – parallel increase of basophils and eosinophils. It can be
observed at allergy latest stages. And if it is so – it is considered as a favorable
prognostic criterium for the patient.
Mast Cell
Mast cell is a large tissue cell resembling the basophil is present in bone marrow and
around the cutaneal blood vessels but does not enter the circulation.
The mast cell plays an important role in producing hypersensitivity reactions like
allergy and anaphylaxy. It secretes heparin, histamine, serotonin, and hydrolytic enzymes.
MONOCYTES – are formed in different hemopoietic organs:
• bone marrow,
• lymphatic nodes;
• connective tissue.
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 Life duration – 36-104 hours. They leave tissues and form macrophagal family there.
Role:
• strong phagocytosis;
• contain monokines influencing on lymphocytes;
• antiinfectional action;
• antitumorogenic activity;
• blood coagulation;
• fibrinolysis;
• complement system components synthesis.
Monocytes play an important role in defense of the body. Along with neutrophils, these
leukocytes constitute the first line of defense. Like neutrophils, the monocytes are also motile
and phagocytic. The monocytes are the free cells in the body and wander freely through the
tissue. Like neutrophils, practically no part of the body is spared by monocytes.
Monocytes secrete interleukin-1 (IL-1), colony stimulating factors (CSF) and platelet
activating factor (PAF).
Monocytes are the precursors of the tissue macrophages. The matured monocytes stay in the
blood only for few hours. Afterwards these cells enter the tissues from the blood and become
tissue macrophages. Examples of tissue macrophages are Kupffer cells in liver, alveolar
macrophages in lungs and macrophages in spleen.

LYMPHOCYTES
The lymphocytes play an important role in immunity. Functionally, the
lymphocytes are classified into two categories namely, T lymphocytes and B
lymphocytes. T lymphocytes are responsible for the development of cellular immunity
and B lymphocytes are responsible for the development of humoral immunity. T-
lymphocytes are thymus-dependent. Their processing in thymus occurs mostly during
the period between just before birth and few months after birth. Thymosin is a
hormone secreted by thymus and released into circulation. Thymosin also plays an
important role in immunity. It accelerates the proliferation and activation of
lymphocytes in thymus. It also increases the activity of lymphocytes in lymphoid
tissues.
There one of their population comes to thymus where their differentiation in T-
lymphocytes takes place. Other part – to bursa of Fabricius analogue (in birds) in small
intestine cellular formations, tonsills, appendix, bone marrow and are differentiated in
lymphocytes (bursa-dependent). The bursa of Fabricius is a lymphoid organs situated
near the cloaca of birds. The bursa is absent in mammals, and the processing of B
lymphocytes takes place in bone marrow and liver. This lymphocytic part is not
differentiated in immune organs and such lymphocytes are called zero-lymphocytes
(neither T-, nor B-).
T-lymphocytes have several types:
• helpers or inducers;
• cytotoxic or killers;
• suppressors;
• memory.

58
 In a whole, they are responsible for cellular immunity. Their amount is 40-70 per
cent of all lymphocytes amount.
Storage of T Lymphocytes
After the transformation, the various types of T lymphocytes leave the thymus,
migrate and stay in the lymphoid tissues present in lymph nodes, spleen, bone marrow
and the gastrointestinal tract.

B-lymphocytes also have several types:

• plasma cells;
• memory cells.
According to another classification – subsets like T-lymphocytes.
They provide immunoglobulins formation (B-lymphocytes are transformed into
plasmocytes – producers of these molecules) and thus delt with cellular and especially
with humoral immunity. Their amount is 20-30 per cent of all lymphocytes.

Storage of В Lymphocyte
After the transformation, В lymphocytes migrate and stay in the lymphoid tissues
present in lymph nodes, spleen, bone marrow and the gastrointestinal tract.

Zero-lymphocytes secrete proteins (perphorines) possessing the ability to make the

foramen in side cells membrane and while protheolytic enzymes (cytolysines) pouring
in them destroy them. That’s why they are often named as natural killers. Their amount
is 10-20 per cent of all lymphocytes.

Leucocytic formula:
• basophils – 0-1,0 %;
• eosinophils – 1,0-4,0 %;
• neutrophils - 50,0-70,0 % - among them:
• juveniles – up to 1,0%,
• rod or stab neutrophils (rods or stabs) – 1,0-4,0 %,
• segs or segment-nuclear neutrophils – 50,0-65,0 %;
• lymphocytes – 25,0-40,0 %;
• monocytes – 2,0-10,0%.
Movement (shift) to the left - is called regenerative movement (blood renewal, sign
of so-called young blood); is characterized by juveniles and rod (stab) neutrophils
increasing in blood. Reasons:
• infectional diseases;
• leucoses;
• inflammational processes.
Movement (shift) to the right - is called degenerative movement (sign of old blood):
is characterized by juveniles and rod (stab) neutrophils amount decreasing and
segment- nuclear leucocytes number increasing. It may be observed at:
• aplastic anemias;
• leucoses.
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 LEUCOPOIESIS REGULATION
Like erythropoiesis regulation it can be performed both specific and non-specific
ways. Specific way – is leucopoietins action (they are produced into liver, spleen,
thymus, kidneys). Their action mechanism is in involving into bone marrow cells
differentiation process. Non-specific way – is:
a) vitamin’s action (especially of groups “B12” and “C”);
b) hormones:
• ACTH;
• thyroid;
• sexual;
c) microelements;
d) leucocytes, tissues, toxin’s, microbes metabolic products have special importance
for leucopoiesis regulation. The more leucocytes are destroyed, the more new forms
are formed.

FIGURE 7: Leucopoiesis

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 The committed pluripotent stem cell gives rise to colony forming unit and lymphoid
stem cell.

COLONY FORMING UNIT

Different colony forming units are:
1. Colony forming unit—Erythrocytes (CFU-E)
2. Colony forming unit—Granulocytes and Monocytes (CFU-GM)
3. Colony forming unit—Megakaryocytes (CFU-M)

DEFINITION AND TYPES OF IMMUNITY

Resistance of the body against the pathogenic agents is known as immunity. It is the
ability of the body to resist different types of foreign bodies like bacteria, virus, toxic
substances, etc. which enter the body. Immunity is of two types namely, innate immunity
and acquired immunity.

INNATE IMMUNITY
Innate immunity is otherwise called natural immunity. It is present from birth and it
is the inborn capacity of the body to resist the entry of microorganisms into the body.
By chance, if the organisms enter the body, innate immunity eliminates them before
they cause any disease. This type of immunity represents the first line of defense
against any pathogens. Therefore, it is also called nonspecific immunity. Some
mechanisms involved in this type of immunity are as follows:
1. Activities of white blood cells and tissue macrophages, which destroy the foreign
bodies by means of phagocytosis
2. The enzymes of gastrointestinal tract and the acid is stomach, which destroy the
toxic substances or organisms entering digestive tract through foods
3. Lysozyme and some polypeptides, which destroy or inactivate the bacteria.

ACQUIRED IMMUNITY
Acquired immunity is the resistance developed in the body against any specific
foreign body like bacteria, viruses, toxins, vaccines or transplanted tissues. So, this type
of immunity is also known as specific immunity. This is the most powerful immune
mechanism that protects the body from the invading organisms or toxic substances.
Lymphocytes are responsible for acquired immunity (Fig. 8).
Main white blood cell function is to participate in defense organism reactions against
foreign agents. There exist the natural (non-specific) and specific defense forms.
The non-specific defense is directed to any foreign agent eliminating. The
phagocytosis, the complement system and others humoral defense factors are the main
types of such reactions. Phagocytosis consists of engulfing the microbes and cells via
the formation of the pseudopods followed by endocytosis of the phagocytic vesicle.
Next, the endocytotic vesicle is incorporated into the lysosomes of the phagocytes
where the microbes and cells are digested by lysosomal enzymes. This phenomenon is
adequate to the neutrophiles, monocytes, eosinophiles, macrophages and
thrombocytes. In the course of the phagocytosis process we differentiate such stages as

61
the phagocyte approaching to the phagocytized object (or ligand), the ligand contact
with the phagocyte membrane, the ligand engulfing, digestion and destruction of the
phagocytized object. The phagocytes find their way to the site of injury by chemotaxis
or similar guiding mechanisms.

FIGURE 8: Schematic diagram of acquired immunity

Complement system - is a special enzyme system consisting of the proteins (more

than 20 types). In includes 9 components (C1…C9). During the activation process
some of its components are cleaved in the fragments influencing directly the course of
specific and nonspecific defense reactions. There exist the classical and alternative
ways of complement system activation. The destruction of foreign and old cells, the
phagocytosis and the immune reactions course activates, the vessel wall permeability
increases, the blood coagulation hastens at the complement system activation that
influence the pathological process.
The other humoral defense factors – defense reactions connected with the action
of such substances as lysozyme and interferon. Lysozyme as a protein possesses the
enzyme activity suppressing the growth and the development of causative agents and
destroying some of the microorganisms. It can be found in nasal mucosa, intestines,
salivary secret, lacrimal fluid etc. In small amounts one can find it in the granules of

62
polymorphonuclear leukocytes, in macrophages and when destroyed they fall into the
extracellular fluid. Interferon as the globulin of blood plasma can be located in the
lymphocytes providing antiviral defense and delaying the cancer cell growth.
Specific defense – immunity – is a reaction complex directed to maintaining the
homeostasis on meeting the host’s body with the antigens which are considered as
foreign (despite their forming in the organism itself or if they come into it from
outside). Under the action of antigen the host body forms the antibodies, activates
lymphocytes and thus they get the ability to participate in the immune response. This
antigen ability to cause the specific immune response is due to the presence of multiple
determinants on its molecule. The active centers of forming antibodies specifically
correspond to the determinants like the key to the lock. The antigen interacting with its
corresponding antigen forms the immune complex.
The immune organs are divided into central (thymus, bursa of Fabricius, bone
marrow) and the peripheral (lymphatic nodes, spleen etc.). There are two categories of
acquired immune responces – humoral or antibody-mediated and cell-mediated.
Acquired immunity developed by the entrance of any foreign body or a vaccine is
called active immunity.
Types of Acquired Immunity
Two types of acquired immunity:
1) Cellular immunity and
2) Humoral immunity
Cellular Immunity
The cellular immunity is by the activation of T-lymphocytes, which destroy the
organisms, entering the body. This is also called cell mediated immunity or T-celled
immunity. This is the major defense mechanism against infections by viruses, fungi and few
bacteria like tuberculosis bacillus. Cellular immunity is also responsible for delayed allergic
reactions and the rejection of tissues transplant from other's body.
Humoral Immunity
Humoral immunity is by the activation of В lymphocytes. This is also called В cell
immunity. В lymphocytes act against the invading organisms by secreting antibodies into
the blood and lymph. The blood and lymph are body fluids (humors) and the В
lymphocytes provide immunity through humors. Therefore, this type of immunity is
called humoral immunity. This plays an important role in defense mechanism against
the bacterial and viral infections.
ANTIGENS
DEFINITION AND TYPES
The antigens are the substances, which induce specific immune reactions in the
body. Antigens are of two types. Those present on the body's own cells are called the
auto-antigens or self antigens. The antigens entering the body from outside are known
as foreign or non-self antigens.
NON-SELF ANTIGENS
Following are the non-self antigens:
1. The receptors on the cell membrane of microbial organisms such as bacteria,
viruses and fungi
2. The toxins from microbial organisms
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 3. The materials from transplanted organs or incompatible blood cells and
4. Allergens or allergic substances like pollen grains.
The non-self antigens are classified into two types depending upon the response
developed against them in the body.
1. The antigens, which induce the development of immunity or production of
antibodies (immunogenicity)
2. The antigens, which react with specific antibodies (allergic reactivity).

CHEMICAL NATURE OF THE ANTIGENS

The antigens are mostly the conjugated proteins like lipoproteins, glycoproteins and
nucleoproteins.
DEVELOPMENT OF CELL-MEDIATED IMMUNITY
INTRODUCTION
The cell mediated immunity is carried by the T lymphocytes. It develops when an
antigen or the antigenic material from the invading microbial or nonmicrobial cells is
exposed to the T lymphocytes. The exposure or presentation of antigen to the
lymphocytes is an important process during development of immunity
(immunogenicity). It is carried out by some special type of cells called antigen presenting
cells.
ANTIGEN PRESENTING CELLS
There are two types of antigen presenting cells in the body.
1. Macrophages and
2. Dendritic cells
1. Macrophages
The macrophages are the large phagocytic cells, which digest the invading organisms
to release the antigen. The macrophages are present along with lymphocytes in almost
all the lymphoid tissues.
2. Dendritic Cells
The dendritic cells are nonphagocytic in nature. Three types of dendritic cells are
involved in this.
1) Dendritic cells in spleen, which trap the antigen in blood
2) Follicular dendritic cells in lymph nodes which trap the antigen in the lymph and
3) Langerhans' dendritic cells in skin, which trap the organisms coming in contact
with body surface.
Role of Antigen Presenting Cells
When foreign organisms invade the body, the macrophages or other antigen presenting
cells kill them mostly by means of phagocytosis. Later, the antigen from the organisms
is digested into polypeptides. The polypeptide products are presented to T lymphocytes
(and also to В lymphocytes) along with human leukocyte antigens (HLAs). HLAs are
the molecules arranged in series in the genes located in short arm of chromosome 6.
The cluster of genes with HLA is called major histocompatibility complex (MHC). HLAs
are easily recognized by the cells of immune systems and hence, the name antigen is given
to them.

64
 Now, the antigenic products activate the helper T cells and В lymphocytes. The
macrophages also secrete some substance called interleukin 1. This causes activation and
proliferation of lymphocytes.
The activated helper T cells are proliferated and released into circulation from
lymphoid tissues.
ROLE OF HELPER T CELLS
Helper T cells help in promoting various activities of immune system. Activated by the
antigenic products of foreign body the helper T cells stimulate the other T cells and the В
cells.
There are two types of helper T cells called T helper-1 (TH1) cells and T helper-2 (TH2)
cells.
Role of TH1 Cells
TH1 cells are concerned with cellular immunity. These helper cells secrete interleukin
2 and gamma interferon Interleukin 2 activates the other T cells. Gamma interferon promotes
phagocytic action of cytotoxic cells, macrophages and natural killer (NK) cells.
Role of TH2 Cells
TH2 cells are concerned with humoral immunity and secrete interleukin 4 and
interleukin 5. These two interleukins are concerned with:
Activation of В lymphocytes
Proliferation of plasma cells
Production of antibodies by plasma cell. Interleukin4 induces the secretion of IgE and
interleukin 5 induces the secretion of IgA.

ROLE OF CYTOTOXIC T CELLS

The activated cytotoxic T cells circulate though blood lymph and lymphatic tissues
and destroy the invading organism by attacking them directly.
Mechanism of Action of Cytotoxic T Cells
The outer membrane of cytotoxic T cells contains some receptor proteins. These
receptor proteins bind the antigens or organisms tightly with cytotoxic T cells. Then, the
T cells are enlarged and release cytotoxic substances like the lysosomal enzymes. These
substances destroy the invaded organisms. Like this, each killer cell can destroy a large
number of microorganisms one after another.
Other Actions of Cytotoxic T Cells
The cytotoxic T cells also destroy cancer cells, transplanted cells like those of
transplanted heart or kidney or any other cells, which are foreign bodies.
Cytotoxic T cells may destroy even the tissues affected by the foreign bodies particularly
the viruses. Man) viruses are entrapped in the membrane of affected cells. The antigen
of the viruses attracts the T cells. And the cytotoxic T cells kill the affected cells also
along with viruses. So, the name killer cell is obtained.

ROLE OF SUPPRESSOR T CELLS

The suppressor T cells are also called regulatory T cells. These T cells suppress the
activities of the killer T cells. Thus, the suppressor T cells play an important role in
preventing the killer T cells from destroying the body's own tissues along with invaded
organisms. The suppressor cells suppress the activities of helper T cells also.
65
 ROLE OF MEMORY T CELLS
Some of the T cells activated by an antigen remain in lymphoid tissue instead of
entering circulation. These T cells are called memory T cells.
In later periods, the memory cells migrate to various lymphoid tissues throughout
the body. When the body is exposed to the same organism for the second time, the
memory cells identify the organism and immediately activate the other T cells. So, the
invading organism is destroyed very quickly. The response of the T cells is also more
powerful this time.
SPECIFICITY OF T CELLS
Each T cell is designed to be activated only by one type of antigen. It is capable of
developing immunity against that antigen only. This property is called the specificity of
T cells.

DEVELOPMENT OF HUMORAL IMMUNITY

INTRODUCTION
Humoral immunity is developed by the antibodies, which are circulating in the blood.
The antibodies are the gamma globulins produced by В lymphocytes. These antibodies
light against the invading organisms. The humoral immunity is the major defensive
mechanism against the bacterial infection. As in the case of cellular immunity, the
macrophages and other antigen presenting cells play an important role in the
development of humoral immunity also.

ROLE OF ANTIGEN PRESENTING CELLS

When foreign bodies or organisms invade, macrophages and other antigen
presenting cells destroy them mostly by phagocytosis. Then, the antigen from the
organisms is digested into polypeptides. The polypeptide products are presented to В
lymphocytes (and also to T lymphocytes) along with human leukocyte antigens
(HLAs).
Now, the antigenic products activate the В lymphocytes and also the helper T
cells. The macrophages also secrete some substance called interleukin 1. This causes
activation and proliferation of lymphocytes.

ROLE OF PLASMA CELLS

The В lymphocytes are proliferated and transformed into two types of cells namely,
plasma cells and memory cells. The plasma cells produce the antibodies, which are
globulin in nature. The antibodies are called immunoglobulins. The rate of the antibody
production is very high, i.e. each plasma cell produces about 2000 molecules of
antibodies per second. The antibodies are released into lymph and then transported into
the circulation. The antibodies are produced until the end of lifespan of each plasma
cell, i.e. from several days to several weeks.

ROLE OF MEMORY В CELLS

Some of the В lymphocytes activated by the antigen are transformed into memory
В cells, which occupy the lymphoid tissues throughout the body. The memory cells are

66
in inactive condition until the body is exposed to the same organism for the second
time.
During the second exposure, the memory cells are stimulated by the antigen and
produce more quantity of antibodies at a faster rate, than in the first exposure. The
antibodies produced during the second exposure to the foreign antigen are also more
potent than those produced during first exposure. This forms the basic principle of
vaccination against the infections.

ROLE OF HELPER T CELLS

Helper T cells are simultaneously activated by the antigen. The activated helper T
cells secrete two substances called interleukin 2 and В cell growth factor, which
promote:
1. Activation of more number of В lymphocytes
2. Proliferation of plasma cells and
3. Production of antibodies

ANTIBODIES
Antibodies or immunoglobulins (Ig) are produced by plasma cells in response
to the presence of antigens. The immunoglobulins form 20% of the total plasma
proteins. Though produced by В lymphocytes, the antibodies are found in
almost all the tissues of the body.
Types of Antibodies
Five types of antibodies are known.
1. IgA (Ig alpha)
2. IgD (Ig delta)
3. IgE (Ig epsilon)
4. IgG (Ig gamma) and
5. IgM (Ig mu)
Among these antibodies, IgG forms 75% of the antibodies in the body.

Structure of Antibodies
Antibodies are gamma globulins with a molecular weight of 1, 50,000 to 9,00,000.
The antibodies are formed by two pairs of chains namely, one pair of heavy or long
chains and one pair of light or short chains. Each heavy chain consists of about 400
amino acids and each light chain consists of about 200 amino acids.
Actually, each antibody has two halves, which are identical. The two halves are held
together by disulfide bonds (S-S). Each half of the antibody consists of one heavy chain
(H) and one light chain (L). The two chains in each half are also joined by disulfide bonds
(S-S). The disulfide bonds allow the movement of the amino acid chains. In each of the
antibody, the light chain is parallel to one end of the heavy chain. The light chain and
the part of heavy chain parallel to it form one arm. The remaining part of the heavy chain
forms another arm. A hinge joins both the arms (Fig. 10).
Each chain of the antibody includes two regions.
1. Constant region and
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 2. Variable region
1. Constant Region
The amino acids present in this region are similar in number and placement
(sequence) in all the antibodies of each type. So, this region is called constant region or
Fc (Fragment crystalline) region. Thus, the identification and the functions of different
types of immunoglobulins depend upon the constant region. This region binds to the
antibody receptor situated on the surface of the cell membrane. It also causes
complement fixation. So, this region is also called the complement binding region.
2. Variable Region
It is smaller compared to constant region. The amino acids occupying this region are
different in number and placement (sequence) in each antibody. So, it is called
variable region. This region enables the antibody to recognize the specific antigen and
to bind itself with the antigen. Because of this, this region of the chain is called antigen
binding region or Fab (Fragment antigen binding) region.

Functions of Antibodies
The functions of the antibodies are:
1. IgA takes part in localized defense mechanism external secretions like tear
2. IgD is involved in recognition of the antigen and represents receptor of lymphocytes
3. IgE is involved in allergic reactions
4. IgG is responsible for complement fixation.
5. IgM is also responsible for complement fixation

Mechanism of Actions of Antibodies

The antibodies protect the body from the invaded organisms in two ways (Fig. 9).
1. By direct actions and
2. Through complement system.

68
 FIGURE 9. Mechanism of immunoglobulins action

1. Direct Actions of Antibodies

The antibodies directly inactivate the invading organ? By any one of the following
methods:
1. Agglutination: In this, the foreign bodies of blood cells or bacteria with antigens
on their surfaces are bound together into a clump by antibodies.
2. Precipitation: In this, the soluble antigens like tetanus toxin are converted into
insoluble forms and then precipitated.
3. Neutralization: During this, the antibodies cover the toxic sites of antigenic
products.
4. Lysis: It is done by the most potent antibodies. These antibodies rupture the cell
membrane of the organisms and then destroy them.
2. Actions of Antibodies through Complement System
The indirect actions of antibodies are stronger than the direct actions and play more
important role in defense mechanism of the body than the direct actions.
The complement system is the one that enhances or accelerates various activities
during the fight against the invading organisms. It is a system of plasma enzymes, which
are identified by numbers from C1 to C9. Including the three subunits of C1 (C1qC1rC1s) there
are 11 enzymes in total. Normally, these enzymes are in inactive form and aге activated in
two ways, namely:
A. Classical pathway.
B. Alternate pathway.
A. Classical pathway

69
 In this the C, binds with the antibodies and triggers a series of events in which other
enzymes are activated in sequence. These enzymes or the by-products formed during
these events produce the following activities:
1. Opsonization: Activation of neutrophils and macrophages to engulf the bacteria,
which are bound with a protein in plasma called opsonin
2. Lysis: Destruction of bacteria by rupturing the cell membrane
3. Chemotaxis: Attraction of leukocytes to the site of antigen antibody reaction
4. Agglutination: By causing clumping of foreign bodies like red blood cells or
bacteria
5. Neutralization: Covering the toxic sites of antigenic products
6. Activation of mast cells and basophils, which liberate histamine. Histamine dilates
the blood vessels and increases capillary permeability. So, plasma proteins from
blood enter the tissues and the antigenic products are inactivated.
B. Alternate pathway
The complementary system can also be activated in another way, which is called
alternate pathway. This is due to a protein in circulation called factor I. It binds with
polysaccharides present in the cell membrane of the invading organisms. This binding
activates C3 and C5, which ultimately attack the antigenic products of invading organism.

Specificity of В Lymphocytes
Each В lymphocyte is designed to be activated only by one type of antigen. It is also
capable of producing antibodies against that antigen only. This property of В
lymphocyte is called specificity. In lymphoid tissues, the lymphocytes, which can
produce specific antibody, are together called the clone of lymphocytes.

NATURAL KILLER CELL

Natural killer (NK) cell is a large granular cell with indented nucleus. It is considered
as the third type of lymphocyte and it is often called the non-T, non-B cell. NK cell kills
the invading organisms or the cells of the body without prior sensitization. It is not
phagocytic cell but its granules contain hydrolytic enzymes. The hydrolytic enzymes play
an important role in the lysis of cells.
NK cell destroys the viruses and the viral infected or damaged cells, which might form
tumors. It also destroys the malignant cells and prevents development of cancerous
tumors. NK cell is said to be the first line of defense in specific immunity particularly
against viruses.
NK cell secretes two cytokines, interferons and tumor necrosis factors.

IMMUNE DEFICIENCY DISEASES

Immune deficiency diseases occur due to lack of some components or some defective
components of immune system. Normally, the defense mechanism protects the body
from invading pathogenic organism. When the defense mechanism fails or defective,
the organisms of even low virulence produce severe disease. The organisms, which take
advantage of defective defense mechanism, are called opportunists. The immune
deficiency diseases caused by such organisms are of two types namely:
1. Congenital immune deficiency diseases
70
 2. Acquired immune deficiency diseases

HLA SYSTEM
In human chromosome 6, there is a series of molecules called human leukocyte
antigen (HLA). The HLA system monitors the immune system in the body. The HLA
molecules are recognized by the T and В lymphocytes and hence these molecules are
called antigens. HLA is distributed in almost all the tissues of the body. The
antibodies are directed against the tissues possessing the HLA leading to autoimmune
diseases. Most of the autoimmune diseases are HLA linked.

IMMUNIZATION
Immunization is defined as the method of preparing the body to fight against a
specific disease. It is a technique used to induce the immune resistance of the body to
a specific disease. It is done by subjecting the individual to an antigen in order to
produce antibodies against that antigen

PASSIVE IMMUNITY
Passive immunity or immunization is produced without challenging the immune
system of the body. This is done bу administration of serum or gamma globulins form
a person who is already immunized (affected by the disease) to a non-immune person.
Passive immunity can bе acquired either naturally (immunoglobulins G can pass
through placenta) or artificially (sera).

ACTIVE IMMUNITY
Active immunity or immunization is acquired by activating immune system of the
body. The body develops resistance against disease by producing antibodies following
the exposure to antigens. Active immunity can be acquired either naturally or artificially.
Active Natural Immunity
Naturally, acquired active immunity involves activation of immune system in the
body to produce antibodies. It is achieved in both clinical and subclinical infections.
Clinical infection: During the disease, the plasma cells produce immunoglobulins
to destroy the invading antigens. Later, due to the activity of memory cells, body
retains the ability to produce the antibodies against the specific antigens invaded
previously.
Subclinical infection: Some times the disease may not be severe to develop any
manifestations. However, it causes the activation of В lymphocytes resulting in produc-
tion of antibodies.
Active Artificial Immunity
This type of immunization is achieved by administration of vaccines or toxoids.
Vaccines consist of dead pathogens or live but attenuated (artificially weakened)
organisms. The toxoids consist of microbial components or toxins secreted by the
pathogens.
CYTOKINES
Cytokines are the hormone like small proteins acting as intercellular messengers by
binding to specific receptors of target cells. These non-antibody proteins are secreted by
71
white blood cells and some other types of cells. Their major function is the activation
and regulation of general immune system of the body.

Depending upon the source of secretion and effects, the cytokines are classified into
five types.
1. Interleukins
2. Interferons
3. Colony stimulating factors
4. Tumor necrosis factors and
5. Chemokines

1. Interleukins
The polypeptide cytokines produced mostly by the leukocytes and exerting the
effects on other leukocytes are known as interleukins (IL). These cytokines are
secreted by helper T cells, other T cells, В cells, monocytes and macrophages. The
actions of interleukins are:
• Activation of T cells, macrophages and NK cells
• Promotion of growth of hemopoietic cells and В cells
• Acceleration of inflammatory response by activating eosinophils and
• Chemotaxis of neutrophils, basophils and T cells
So far, about 16 types of interleukins are identified. IL-1, IL-2, IL-3, IL-4, IL-5, IL-
6, and IL-8 play important role in the process of immunity. Recently IL-12 (otherwise
called natural killer cell stimulatory factor) and IL-II also considered as an important
cytokines.

2. Interferons
The interferons (IFN) are the glycoprotein molecules set produced by white blood cells,
natural killer cells and it blasts. Considered as antiviral agents, these cytokines have the
following effects:
1. Fighting against the viral infection by suppress the virus multiplication in the target
cells
2. Inhibition of multiplication of parasites and cells
3. Promotion of phagocytosis by monocytes and macrophages
4. Activation of NK cells. Interferons are of three types namely, INF-б, INF-Я and
INF-г.

IMMUNODEFICIENCY is a failure of some part of the immune system to

function properly. It can be congenial (present at birth) or acquired. Congenital
immunodeficiencies usually involve failure of the fetus to form adequate numbers of
B cells, T cells or both. Severe combined immunodeficiency (SCID), in which both
T cells and B cells fail to form, is probably the best known. Unless the person suffering
from SCID is kept a sterile environment or is provided with a compatible bone marrow
transplant, death from infection results.
Acquired immunodeficiency can result from many different cases. For example,
inadequate protein in the diet inhibits protein synthesis and, therefore, antibody level
72
decrease. Immunity can be depressed as a result of stress, illness, or drugs such as those
used to prevent graft infection. Diseases such as leukemia cause an overproduction of
lymphocytes that do not function properly.

ACQUIRED IMMUNODEFICIENCY SYNDROME (AIDS)

It is a life-threatening disease caused by the human immunodeficiency virus (HIV).
Two strains of HIV have been recognized: HIV-1 is responsible for most cases of
AIDS, whereas HIV-2 is increasingly being found in West Africa. AIDS was first
reported in 1981 in the United States. Since then over 500000 cases have been reported
to the Center for Disease Control (CDC). Evidence suggests that almost everyone
infected will develop symptoms within 10 years, and they will eventually develop the
disease if they do not die of some other cause. The few causes of HIV-infected
individuals who have not developed AIDS even after many years of being infected are
being investigated.
HIV is transmitted from an infected to a non-infected person in body fluids such as
blood, semen, or vaginal secretions. The major method of transmission are intimate
sexual contact, contaminated needles used by intravenous drug users, and tainted blood
products. Present evidence indicates that household, school, or work contacts do not
result in transmission.
In the USA, most cases of AIDS have appeared in homosexual or bisexual men are
in intravenous drug users. A small percentage of cases have resulted from transfusions
or contaminated clotting factors used by hemophiliacs. Sadly, children can be infected
before birth, during delivery, or after birth from breast-feeding. A few cases of AIDS
have occurred in health care workers accidentally exposed to HIV-infected blood or
body fluids, and even smaller number of cases of health care workers infecting patients
has been documented. The most rapidly increasing group of AIDS patients in the
United States of America is heterosexual women or men who have had sexual contact
with infected person. Women in the 15- to 25-year-old age group appear to be
especially likely to contract AIDS, possibly because the vaginal mucous membranes
of women in this age group are thin and are a less effective barrier to the virus.
In the other countries, the pattern of AIDS cases can be different from that in the
USA. For example, in Haiti and central Africa, heterosexual transmission is the major
route of HIV spread. Worldwide, about 40% of AIDS are women. The World Health
Organization estimates that near 27 million people have been infected by HIV.
Preventing transmission of HIV is presently the only way to prevent AIDS. The risk
of transmission can be reduced by educating the public about safe sexual practices such
as reducing the number of one's sexual partners, avoiding anal intercourse, and using
condom. Public education also includes warnings to intravenous drug users of the
dangers of using contaminated needles. Ensuring the safety of the blood supply is
another important preventive measure. In April, 1985, a test for HIV antibodies in
blood became available. Heat treatment of clotting factors taken from blood has also
been effective in preventing transmission of HIV to hemophiliacs.
HIV infection begins when the virus binds to a CD4 surface molecule. The CD4
molecule is found primarily on helper T cells but also on certain monocytes,
macrophages, neurons and neuroglial cells. Once attached to CD4 molecule, the virus
73
injects its genetic material (RNA) and enzymes into the cell. The viral RNA and
enzymes produce DNA that can direct the formation of new HIV ribonucleic acid and
proteins, that is, additional viruses that can infect other cells. Most of the manifestations
of AIDS can be explained by the loss of helper T cell functions or the infection of the
other cells with CD4 molecules. Without helper T cells, cytotoxic T-cell and B-cell
activation is impaired, and specific resistance is suppressed.
Following infection by the HIV, within 3 weeks to 3 months, some patients develop
an acute (sudden0 mononucleosis-like syndrome that can last up to 14 days. Symptoms
include fever, sweats, fatigue, muscle and joint aches, headache, sore throat, diarrhea,
rash, and lymphadenopathy. More commonly there is a persistent version of the
syndrome that lasts for several months and includes lymphadenopathy, fever and
fatigue. During this time the patient becomes positive for HIV antibodies, and within
a year many patients develop AIDS.
The most common clinical manifestations of AIDS include testing positive for HIV
antibodies, a decrease in helper T cell numbers to fewer that 200/mm 3 of blood, and
the presence of opportunistic infections or Kaposi's sarcoma. Normally, there are about
1200 helper T cells/ mm3 of blood, but between the time of infection and the AIDS
development the T-helpers decreases. Apparently most HIV replication takes place in
the lymph nodes where helper T cells and other immune cells aggregate. As cells in the
lymph nodes are destroyed, the number of circulating helper cells decreases.
Opportunistic infections involve organisms that normally do not cause disease but
can do so when the immune system is depressed. Examples include Pneumocystis
carinii (pneumonia caused by intracellular protozoan); tuberculosis (caused by
tuberculosal Mycobacter), syphilis (caused by pale Treponema), candidiasis (a yeast
infection of the mouth or vagina caused by Candida albicans); and protozoans can
cause severe, persistent diarrhea. Kaposi's sarcoma is a type of cancer that produces
lesions in the skin, lymph nodes, and visceral organs. Also associated with AIDS are
symptoms resulting from the effects of HIV of the nervous system, including motor
retardation, behavioral changes, progressive dementia and possibly psychosis.

DIFFERENTIATED LEUCOCYTES AGEING CHANGING IN CHILDREN

Differentiated leucocytes is significantly changed while ageing. Both mature and
immature children have such changings beginning from the 2-nd day of life:
neutrophils number decreasing (neutrophilopeny) and lymphocytes number increasing
(lymphocytosis). These cellular elements number got equal to the 5-6th days (in the
mature) and to the 3rd day (in the immature). It is so-called the first crossing. Segments
minimal numbers and lymphocytes maximal numbers are determined in the age of 5-6
months in the mature and in the age of 1-2 months in the immature. Then neutrophils
are getting increased while lymphocytes – decreased. Their number got equal again at
4-5 years (the second crossing). Direction into neutrophils and lymphocytes number
changing is remained non-changed up to 14-15 years when these cellular elements
content get like in the adult.
Both the mature and the immature have myelocytes and metamyelocytes in blood
during the first 2 weeks of life. Monocytes number is rather low right after birth in the
mature, then (during the next 2 weeks) their number get increased (in average up to
74
10,5%). Further changings are the following: decreasing during the first year of life up
to 7-8%, then – up to 6%. Non-mature children have the same tendency but absolute
numerals are less. Eosinophils content is relatively higher in the new-borns than in next
age periods.

LEUCOCYTES FUNCTIONS SIGNIFICANCE IN DENTISTRY

There are more than 400 microorganisms specieces in human mouth. Some of them
can be the reason of gums infectionning and the one of sublaying bone tissue.
Favourable conditions in oral cavity for microorganisms are as follows as:
• food residues;
• saliva weak-alkaline character;
• humidity;
• optimal temperature.
Microorganisms represent near 70% of dental covering: dental covering dry mass 1
ml contain up to 250 microbial cells; in 1 ml of saliva – more than 108 of microbes.
Viruses and bacteries distribution is unequal in oral cavity.
The microbes biggest amount is in:
• odontal-gingival pockets;
• mucosal plicas;
• interdigital spaces.
Pathogenic microflora plays essential role at gums injury in part at parodontosis that,
as it is well-known, leads to teeth loosing.
Oral microflora has relative stability mainly due to bacteriostatic and bacteriocydic
features.
Main salivary compounds that have defective qualities:
1) lysozyme (muromidase);
2) lactoperoxidase;
3) myeloperoxidase;
4) mucin;
5) beta-lysins;
6) interpherons;
7) proteolyitc enzymes with wide activity spectrum;
8) lithium ions;
9) cyanides;
10) secretory lg A (SlgA);
11) serum lg A, G, E;
12) complement system components C3 and C4.
Lysozyme bacteriolytic action deals with muramic acid decomposition in some
bacteries walls as a result of which its permeability is changed and its content diffunds
in surrounding environment.
Lactoperoxidase makes bacteriocydic action – participates in gram-negative
bacteries lysis – due to:
• bacteriocydic aldehydes formation;
• strong oxidizers (galogens) insertion in bacterial membrane.

75
 Myeloperoxidase – encouraging lipids peroxidative oxidation and leading to
bacteries death.
Lactoferrin – make a competition with bacteries for Fe ion and, if bacteries have
developed cytochrome system, - leads to their death.
Mucin – encourages bacteries attachement to desquamating epitheliocytes. Oral
liquid washes desquamating cells and they are digested in stomach after saliva
swallowing. Simultaneous bacterial death occurs in an acidic gastric environment.
Beta-lysines – penetrate into oral cavity from blood by passive diffusion. They
influence on bacteries membrane leading to their death.
Interpherons –are present in saliva and make antiviral, immunomodulative,
radioprotective and antitumorogenic qualities.
Proteolytic enzymes – are capable to damage some bacteries membranes.
Lithium ions, cyanides – their presence also leads to microorganisms death.
Secretory lg A content is higher in saliva than in blood serum. Submucosal layer
contains much B-lymphocytes that are transformed into plasmocytes under interleukins
action (IL are released by T-helpers of the II-nd class). Slg A main features are:
• unsensitivity to salivary proteases;
• unsensitivity to bacterial proteases.
SlgA are capable to bind bacteries exotoxins.
Ways of antibacterial activity:
• bacteries agglutination;
• their reproduction limiting;
• prevention of their attachment to epithelium.
Also SlgA possesses expressed virus-neutralizing activity.
SlgA deficiency in oral cavity is accompanied by often inflammatory diseases.
Serum lg G, A and E – prevent infectious diseases development. lg G can be
produced in little concentrations with oral mucosa plasmocytes; lg E and A passes into
saliva from blood by passive diffusion.
Complement system subfraction C3 and C4 – play important role into:
• phagocytosis activation;
• T- and B-immunity stimulation.
It is proposed that complement system components come into saliva from blood
stream through odontal-gingival sulcus.
Barriers play important role in protection from pathogenic microorganisms, toxins
and other injuring agents. They encourage organs and tissues cells prevention from
contact with damaging agents. Oral mucosa epithelium has such a function in oral
cavity.
Tongue possesses especially powerful barrier function because it is covered by
keratinizing multi-layered epithelium while gums are covered by keratinizing one-
layered epithelium that is why their barrier function is expressed relatively weak. But
this deficiency is compensated by many phagocytes in gums. Phagocytes are located
directly in submucosal layer. Besides, connective tissue contains antibodies where,
probably, they are produced by local plasmocytes. In part, one can find lg M, G and A
in gingival mucosa.

76
 If salivary components and tissular barrier can not perform protective functions
properily and there is a possibility to get sick for organism than non-specific resistance
and immunity mechanisms are involved in defense with microflora. Lymphoid tissue
located in oral cavity (Pirogov-Langhans' ring) in part palatinal and lingual tonsils
play important role in these reactions. Processes that take place in tonsils are as follows:
• toxins partial detoxication;
• viruses neutralization;
it is a “home” for T- and B-lymphocytes: when migrated in oral cavity lymphocytes
are capable to be destroyed with lyzosomal enzymes that damage pathogenic
microorganisms membranes.
Phagocytosis plays essential role in oral cavity protection but its action is realized
only under pathological conditions.
It is known that leucocytes death takes place:
• directly in tissues;
• in spleen.
About 40 billions of leucocytes die daily. These cells significant part is released from
blood to mucosae surface and in oral cavity especially. There are such data that near
350-370 mln of L id est 1/80 of all WBC in vascular bed are released into oral cavity
only from gums blood. This number is increased in 2-10 times at inflammation. Under
physiological conditions 1 mm3 of saliva contains up to 600 leucocytes.
Leucocyte formule of saliva and parodontal pockets gingival liquid:
• 95-97% - neutrophils;
• 1-2% - lymphocytes;
• 2-3% - monocytes.
Oral liquid neutrophils in a healthy person do not possess phagocytic activity. But
they release enzymes that are capable to influence on oral mucosae as well as on
microorganisms here. At the same time leucocytes exhibit expressed phagocytic
activity at inflammation and traumas in oral cavity.
It is important to mention that the old with adenty washing from oral liquid does not
contain any WBC because they migrate only from gums margins surrounding teeth.
It has been recently established that saliva has compound partially protecting
lymphocytes from AIDS virus penetration into them. It is known that saliva encourages
viral particles agglutination. Probably, factor providing virus agglutination is a protein
that is different from mucin by its features. This protein is received now and purified
by American scientists. It took the name “fusin” (fusion – binding to smth). Its
technology has been worked out and it was applied successfully for treatment the
patients with lungs pathology as well as in dentistry. Fusin is synthesized by salivary
glands cells. It binds to leucocytes membranes and thus protects them from virus
passage into WBC. It was proposed that this protein inhibited those molecules of
membrane that helped virus passage through membrane. Fusion or viral and cellular
membranes merging appears in a process of virus passage into the cell. Fusin is similar
to chemokine by its content and functions. Chemokines represent biologically-active
substances that mediate chemotaxis, phagocytosis, cellular and humoral immunity.

77
 Protective cells.
Oral mucosa epithelium serves as a barrier in the way of antigens penetrations in
part allergens, cancerogens as well as microorganism invasion. Epithelium barrier
function disorder leads to oral cavity multiplied diseases appearance. At the same time,
leucocytes mostly degeneratively changed neutrophils are found in mucosal epithelium
as well as on its surface. Separate neutrophils make microorganisms phagocytosis and
probably represent antigen-presenting cells (APC).
Neutrophils and monocytes big amount is under epithelium through which they
migrate from proper plane vessels in gingival sulcus lumen. Neutrophils migration
speed is 30000 cells per 1 min and their relative portion in epithelium is equal to 60%.
T-lymphocytes mainly T-helpers of the I-st and the II-nd classes can be found in oral
mucosa epithelium. Up to 40% of T-lymphocytes are in movement. They can be
located in groups and separately. Interepithelial lymphocytes are undergone to
apoptosis in many focuses. Their big part acquires phenotype of memory cells and
participates in secondary immune response.
Langerhans' cells play important role in oral cavity epithelium barrier function
providing. They comprise 2% of cellular population. They are similar to epidermis
similar cells by their morphological-functional characteristics. They are mostly in
constant movement. It facilitates their meeting with antigens. These are real antigen-
presenting cells (APC). They migrate in regional lymphatic nodes after meeting
antigens where not only contact and side agent transduction to lymphocytes takes place
but the proliferation of the latest ones occurs as well. Dendritic APC with phenotype
CD36 are in oral mucosa epithelium similar to macrophages by their ultrastructure.
Epitheliocytes, lymphocytes and Langerhans' cells interaction
Epitheliocytes start IL-1 and TNF-alpha (tumor-necrotic factor) production after
antigen meeting. It causes Langerhans' cells stimulation. These cells are able to secrete
IL-1 and IL-6 acting to T-helper of the I-st subclass after contact with antigen and
antigen transformation into immunogenic form. T-helpers of the I-st subclass secrete
IL-2 and gamma-interferon that leads to APC activation. At T-helpers of the II-nd
subclass involvement into immune response B-lymphocytes transformation into
plasmocytes occurs capable to produce lg. Immunoglobulins can be both in free and
bound form in oral mucosa epithelium. Free lg are present in: serum, lymph and tissular
liquid. Bound lg form immune complexes with antigens that are eliminated by
phagocytes. Ig A and lg G are found in mucosa the most often, while lg M – at
inflammation. Intraepithelial lg take part both in antigens elimination and in
inflammation process.

4. Materials for auditory self-work

4.1. List of study practical tasks necessary to perform at the practical class.
Materials and methods: microscope, mixer (melanger less than erythrocytic one) for
leucocytes counting, Goryaev’s chamber, blood, hemocytometers.
Investigative object: human being.
Leucocytes amount as erythrocytes one may be estimated both rutine (non-
automatical) method (estimation in Goryaev’s chamber) and automatical methods.

78
 Task 1
Leucocytes estimation in Goryaev’s chamber
It’s necessary to petch blood in melanger for leucocytes till the mark 0,5 or 1,0. To
wipe melanger’s end with cotton wool and then, putting it in dilutor – 5% acetic acid,
colored with methylenic blue, to petch melanger’s content. To pour the first 1-2 blood
drops onto cotton wool, to fill up chamber with the third one. The investigator must
estimate leucocytes under small increasing in 25 large (400 small) squares. Leucocytes
amount is estimated on formula:

X=(a x 4000 x 20 or 10) : (25 x10) x 106,

where:
X – leucocytes amount in 1 l of investigated blood;
a – leucocytes amount determined in course of count;
4000 mcl/mm3 – small square volume;
20/10 - blood dilution degree;
400 – small squares amount;
106 – re-count co-efficient in international units system.

5. Literature recommended:
1. Lecture course.
2. Mistchenko V.P., Tkachenko E.V. Methodical instructions for dental students
(short lecture course).-Poltava, 2005.-P.39-41, 45-46.
3. Mistchenko V.P., Tkachenko E.V. Methodical instructions for medical students
(short lecture course).-Poltava, 2005.-P. 66-69.
4. Mistchenko V.P., Tkachenko E.V. Blood system Physiology //Methodical
recommendations to practical classes for students of medical and dental
departments.-Poltava, 2005.-20p.
5. Kapit W., Macey R.I., Meisami E. The Physiology Colouring Book: Harpers
Collins Publishers, 1987.-P. 139-140.
6. Guyton – Ganong – Chatterjee. Concise Physiology /Ed. By Dr Raja Shahzad
Gull: M.B.B.S., F.C.P.S., King Edward Medical College.-Lahore, 1998 (1st
Edition).-P.178-192, 205-208.
7. Stuart Ira Fox. Human Physiology.-8th Ed.-McGrawHill, 2004.-P.369-371, 444-
477.
8. Seeley R.R., Stephens T.D., Tate P. Essentials of Anatomy and Physiology.-The
3rd Ed.-McGraw Hill, 1999.-P.291-293, 372-392.

6. Materials for self-control:

1. Control questions.
2. Leucocytes classification.
3. Leucocytic formula.
4. Separate leucocytes functions.
79
 5. Leucopoiesis and its regulation.
6. Leucocytes functions significance in dentistry.

LESSON 35
PLATELETS AND VASCULAR-PLATELET HEMOSTASIS
INVESTIGATION
1. The topic studied actuality.
Haemostatic reactions are relatively constant during human life. Such reactions are
provided by neuro-endocrine regulation complex mechanism as well as organs
producing both activators and inhibitors of this process.
Important role in such a complicated regulatory system play salivary glands
secreting and releasing with saliva in oral cavity haemostatic factors influencing on its
vascular-platelet and coagulational-fibrinolytic link.
Particularly, saliva influences on platelets aggregation. Under its influence: latent
period and aggregation velocity are accelerated, aggregates become bigger. Saliva also
influences on platelet thrombus and fibrin clot retraction. It testifies to existence in it
such substances like thrombostenin. Besides, this process acceleration can also be delt
with calcium existence in saliva. Finally, aggregation can be caused by thromboxane
B2 which is one of pro-aggregators (it is more powerful than thromboxane A2 though
it has less time period comparatively to A2).
Saliva influence on platelets aggregation can also depend on antiaggregational
features of salivary glands and parodont tissues. Parodont possesses mainly
proaggregatory features.
Correlation between these substances (of anti- and proaggregational nature) in
parodont, salivary glands, oral mucosa, saliva will define microcirculative haemostasis
in a given part of alimentary tract. At proaggregational features enforcement
microcirculatory hemostasis disorder can occur in mentioned substrates. Such
enforcement will encourage parodontitis, syaloadenites and will influence also on
interrelations between oral mucosa tissues and denturing materials in course of
orthopedic treatment.

Complications after teeth extraction in patients with microcirculative

hemostasis disorders
In patients with platelet-vascular hemostasis disorders (thrombocytopenias) one can
register cases of abundant and prolonged bleedings from alveole of extracted tooth.
Such bleedings character and duration are determined by local (volume and degree of
tissues injure) and general (vessels and homeostasis diseases) reasons.
Aspirin and other non-steroid anti-inflammatory medicines belong to rather widely-
spread practically in all branches of medicine. That is why doctors must know further
information about them. Aspirin inhibits the cyclooxygenase enzyme that catalyzes
the conversion of arachidonic acid (a cyclic fatty acid) into prostaglandins, and thereby

80
inhibits the release reaction and consequent formation of a platelet plug. Since platelets
lack nuclei and are not complete cells, they cannot regenerate new enzymes. Therefore,
the enzymes remain inhibited for the life of the platelets. The ingestion of excessive
amounts of aspirin can thus significantly prolong bleeding time for several days, which
is why blood donors and women in the last trimester of pregnancy are advised to avoid
aspirin. Sight inhibition of platelet aggregation by low doses of aspirin, however, can
reduce the risk of atherosclerotic heart disease, and such a regimen is often
recommended for patients diagnosed with this condition. Because aspirin inhibits
prostanglandin synthesis, the production of thromboxanes, which are derived from
prostaglandins, is also inhibited, resulted in reduced platelet activation. If an expectant
mother ingests aspirin near the end of pregnancy, prostanglandin synthesis is inhibited,
with several effects. Two of these effects are:
1) the mother experiences excessive bleeding after delivery because of decreased
platelet function;
2) the baby can exhibit numerous localized hemorrhages over the surface of its
body as a result of decreased platelet function.
If the quantity of ingested aspirin is large, the infant, mother, or both may die as a
result of bleeding.
On the other hand, in a heart attack or stroke, platelet plugs and clots can form in
vessels and be life-threatening. Studies of individuals who are at risk from the
development of clots, such as people who had a previous heart attack, indicate that
taking small amounts of aspirin daily can reduce the likelihood of clot formation and
another heart attack. It is not currently recommended, however, that everyone should
take aspirin daily.
Primary (vascular-platelet) hemostasis disorders are the reason of almost 80% of
bleedings and 95% cases of thromboses.
Hemorrhagic syndrome can be present is a patient of any age. But this problem is
the most actual in pediatry because significant amount of innate and acquired
hemorrhagic diseases have early expression. Rate of thrombocytopathies (diseases
with platelets dysfunction) are rather spread in a human population. For example, fon
Willebrandt disease (innate adhesive thrombocytopathy) rate is fluctuated from 1/800
to 1/500 and is inherited by autosome-recessive way. Fon Willebrandt factor, adhesion
agent, releasing is disturbed at this disease. Scientists deal this pathology with the 12 th
chromosome where the gene of mentioned factor is located.

2. Study aims:
To know: platelets structure, functions, number, vascular-platelet hemostasis
mechanism, its importance in general medicine and dentistry, thrombocytopoiesis and
its regulation.
To be able to: to determine bleeding time (by Duke), to perform aggregatogram
analysis and to assess the indexes received.

3. Pre-auditory self-work materials.

3.1.Basic knowledge, skills, experiences, necessary for study the topic:

81
 Subject To know To be able to
Biology and Inheritance main ways Analyze primary
Medical thrombocytopathies inheritance
Genetics ways
Pathophysiology Vascular-platelet hemostasis Interpret data received about
reasons, disorders platelets amount, adhesion,
classification, ethiology, aggregation (in part aggregatogram),
pathogenesis, clinics, blood bleeding time by Duke
analysis changes
Pediatry with Platelets and vascular-platelet Say about thrombocytopenies and
Neonatology hemostasis peculiarities in thrombocytopathies in children
children of different age,
thrombocytopoiesis and its
regulation
Internal Platelets structure, function, Tell about ethiology, pathogenesis,
Diseases number, vascular-platelet treatment and prophylaxy of primary
hemostasis mechanism, its hemostasis disorders
importance in general
medicine
Surgery About ethiology, To prevent and to liquidate
pathogenesis, treatment and complications after bleedings in
prophylaxy of primary patients with microcirculative
hemostasis disorders hemostasis disorders
Dentistry Oral cavity organs role in To prevent and to liquidate
primary hemostasis, complications after teeth extraction
complications after teeth in patients with microcirculative
extraction in patients with hemostasis disorders
microcirculative hemostasis
disorders

Topic content
Platelets are 2 – 4 µ in diameter, volume is 6-9 mcm3; they are the smallest blood
cells. They are developed from giant cells called megakaryocytes.

82
 FIGURE 10. Platelets in blood smear.
FIGURE 11. Intact and activated (with pseudopodias) platelets

THROMBOCYTES STRUCTURE
◼ Spherical, oval, or rod-shaped colorless bodies.
Diameter is between 2 to 4 м. They are activated in course of side surface, many (up
to 10) processes are appeared in them as a result of which their diameter is increased
in 5-10 times. There are integrines on platelet membrane serving as receptors. They
participate in thrombocytes interaction one to another and to injured vessel. They have
glycoprotein nature expressing fibrinogen, collagen, Willebrandt factor (FW) and other
substances.
◼ When unstimulated, under Electron Microscopy they appear as:
• Flattened discs
• Having a cell membrane
• And a Cytoplasmic matrix.
• Microtubules encircle the thrombocyte just below it’s surface membrane.
◼ Do not have nuclei.
◼ Can not reproduce.
◼ But behave functionally as whole cells.
◼ Cytoplasm includes active proteins such as:
• Actin.
• Myosin.
• Thrombasthenin.
◼ Cell Organelles in thrombocytes include:
• Lysosomal granules.
• Dense bodies: about 50 – 100 in number.
• Mitochondria & Enzyme systems which produce:
1) ATP
2) ADP
• Enzyme systems producing:
1) Prostaglandins – Local hormones.
• Fine Glycogen granules.
83
 ◼ Microvesicles.
◼ Microtubules.
◼ Filaments.
◼ Granules.
◼ Internal Membranous systems:
• Open Canalicular System:
1) Sponge-like invaginations
2) Provide multiple channels for:
a) Taking up Calcium ions
b) Secreting granule contents.
• Dense Tubular System:
1) Channels of S.E.R.
2) Serves as an intracellular store for Calcium ions.

PLATELETS PHYSIOLOGY
• Have a half life of 8 – 12 days.
• Eliminated from circulation by the Tissue Macrophage system.
• Thrombocytes are active structures.
• About half of them are removed by the Macrophages in the Spleen.
• Platelet surface membrane has Phospho lipids, Cholesterol & Glycolipids
Platelets contain many granules with great number of biologically active substances.
One can differentiate:
1) Alpha-granules – contain more than 30 proteins delt with haemostasis and other
reactions:
• platelet factor 4;
• fibrinogen;
• thrombostenin and so on.
2) Dense granules – they contain biologically active substances delt with vascular tone
and hemostasis:
• ADP;
• epinephrin;
• serotonin;
• thromboxans et al.
3) Lysosomal granules:
• kinases;
• enzymes.

Platelet number under norm is:

• in adults - 180-320 x 109/l; (boardering – 150 – 400 x 109/l);
• in new-borns - 200 x 109/l (in average – fluctuations 100-400 x 109/l);
• 7-10th days – 150-200 x 109/l;
• 14th day – like in adulthood;
• 1-5 years: boys – 217-407 x 109/l, girls – 229-553 x 109/l;
• 10-15 years: boys – 156-408 x 109/l, girls – 154-442 x 109/l;
• 16-20 years: guys – 140-392 x 109/l, girls – 154-386 x 109/l.

84
 4 forms of platelets:
• junior – 0-0,8%;
• mature – 90,3-95,1%;
• old – 2,2-5,6%;
• degenerative – 0-0,2%;
• irritation forms – 0,8-2,3%.

AMOUNT CHANGING
Thrombocytosis (more than 500 x 109/l) - their amount increasing:
1) Physiological:
• pain (noceoceptive);
• stressogenic;
• muscular (in course of physical loading).
2) Pathological:
• spleen pathology;
• spleen removal.

Thrombocytopenia (less than 140 x 109/l) – as a rule, is pathology sign and is

observed at:
• radiation disease;
• congenital blood diseases;
• acquired blood diseases.
But in women on course of menstruation period platelets amount may be reduced
though they are seldom out of normal limits. But it should be mentioned that even at
strong thrombocytopenia reaching up to 50 x 109/l, there is no any bleedings and
women mustn’t be under medication at these situations. Only when reaching critical
ziphras – 25-30 x 109/l - light bleedings may occur at which treaty measures are
essential. It testifies that platelets amount in blood is excessive.

PLATELETS FUNCTIONS:
1. Participation in vascular-thrombocytic and coagulational haemostasis (properly
blood coagulation).
2. Angiotrophyc function – vascular walls feeding (15 per cent of all thrombocytes
daily). At this function disorders: vascular wall permeability increasing and
resistance decreasing.
3. Protective function:
• phagocytosis;
• contain immunoglobulins;
• lysozyme source;
• they are essential for reparation;
• cytokines source.

THROMBOCYTOPOIESIS REGULATION
1. Specific:
• thrombocytopoietins;
• interleukines-3,6,7,9,11,13.
85
2. Non-specific:
a) hormones:
• adrenocorticothropic;
• adrenaline;
b)food products:
• nettle;
• fungus puff-ball;
c) sympathetic nervous system excitement.

HEMOSTASIS
Hemostasis – is the reactions complex aimed at the blood loss stopping as well as
blood liquid state maintaining in vascular bed. Hemocoagulation – hemostasis system
part directed to bleeding stoppage. But here we will use the term “hemostasis” in its
narrow aspect – in meaning of hemocoagulation. In fact the significance of hemostasis
system is much more complicated and far exceeds the limits of fighting with blood
loss.
The main tasks of the hemostasis are the following: the fluid blood state storage, the
transcapillary exchange, the vessel wall resistance regulation and the influence on the
reparation processes and so on.
They distinguish the vessel-platelet hemostasis and blood coagulation (clotting).
Speaking about the first case the question is about the blood loss stopping from the
small vessels with low blood pressure; the second one is connected with the blood loss
fighting at the arteries and veins rupture. Such a division is rather conditional as both
at small and large vessels rupture together with the thrombocyte plug forming the blood
coagulation is occurred. On the other hand such a division is very suitable for clinical
practice because at the vessel-platelet hemostasis disorders the finger skin puncture (or
the ear lobe) is accompanied by prolonged coagulation time whereas the bleeding time
remains normal (for example at haemophilia because of normal platelet count in
hemophiliac). Hemophilia is a wide-spread hereditary pathological state. It is the
excessive bleeding caused by a congenital lack of a substance (plasma coagulation
factor VIII, IX, X or XI) necessary for blood clotting. Treatment consists of
administration of the deficient factor.
Vessel-platelet or vascular platelet hemostasis (or so-called primary one) comes
to the platelet plug (or thrombus) forming.

PLATELET PLUG FORMATION

• It can by itself stop blood loss, if the rent is small.
• Many such minute ruptures occur thousands of times every day in minute blood
vessels.
• Platelets manage to plug these very well, all by themselves.

Vascular-platelet hemostasis
The 1-st stage
vessels temporary spasm:
Vessels injury
86
 ADP
collagen Vessels primary spasm
epinephrine
thromboxan A2

Epinephrine and norepinephrine platelets

increasing in blood activation

The 2-nd stage Glycoproteids expression

Platelet plug formation

platelets ADP Platelets fibrinogen

adhesion aggregation
(primary,
Willebrand’s platelets
reversible)
factor activation
factor
thrombin
fibronectin

Releasing reaction

Lysosomes: б-granules: optically-densed

kinases, factor 4, granules:
enzymes fibrinogen, ADP,
thrombosteninne epinephrine,
serotonin,
thromboxane A2,

Platelets aggregation (repeated, irreversible)

The 3rd stage: Vessels secondary spasm

platelet plug retraction Retraction

87
 Scheme 1. Vascular-platelet haemostasis
The first stage is temporary (primary and secondary) vasoconstriction -
immediately in a few seconds after the injury the primary vasoconstriction occurs due
to it the bleeding at the first moment may not happen or bears the limited character. It
is caused by the adrenaline or norepinephrine releasing in response to the pain irritation
and lasts for about 10-15 sec. Futher, the secondary vasoconstriction occurs because of
the platelet activation and the releasing from them in a blood the vasoactive substances
- serotonin, adrenaline, thromboxanes.
The second stage is the platelet plug forming because of the adhesion (the binding
to the foreign surface) and the aggregation (clumping of the platelets). The adhesion
takes place immediately after the injury to the collagen and other adhesion
subendothelium proteins. It occurs because of the glycoproteins action by means of
which the platelets clump to the collagen fibres and by means of the Willebrand factor
as well that one of its active centers usage is bound up to the platelet receptor and the
other of its receptors to the collagen or subendothelium. From the adhesive platelets
and the injured endothelium as well the ADP (adenosine diphosphate) is released,
which is one of the major factors of platelet aggregation. Under the unfluence of ADP
the platelets clump, so forming the aggregates. This reaction increasing is due to the
platelet activation factor (PAF), thrombin and adrenaline. On this stage the aggregation
is reversible and the desaggregation may happen. To complete the platelet plug
forming a number of additional mechanisms (they are associated mainly with the
platelets) are required. When the signal comes into the platelets the calcium content
increases in them and the phospholipase A2 activation occurs. The latter one leads to
the arachidonic acid releasing from the platelets membranes that further converts into
the very active prostaglandines and thromboxanes. When removing from the platelets
they make the aggregation irreversible. As a result the platelet plug or thrombus is
formed. But at first it is capable of passing the blood as it is loose. After releasing the
actomyosine (thrombostenine) from the platelets during their aggregation the platelet
plug is shortened and reinforced. This is the third stage of the vessel-platelet
haemostasis – the platelet plug retraction.
Under the normal condition the blood loss stoppage from small vessels lasts from 2
to 4 minutes. Such index in the clinic is known as the bleeding time.
The arachidonic acid derivates – prostacyclin and thromboxane A2 - play a very
important role in the vessel-platelet haemostasis regulation. Prostacyclin is produced
by endotheliocytes under the enzyme prostacyclinsynthetase influence. Under the
physioilogical conditions prostacyclin predominates over thromboxane – powerful
platelet proaggregant. At any endothelium injury in the trauma place the prostacyclin
producing disturbs and the thromboxane action begins to be predominate. Thus, the
favourable conditions for the platelet aggregation emerge. Some vitamins (A,C,E) and
foods (onion, garlic) are the platelet aggregation inhibitors.
Blood coagulation is an enzyme process where both the plasmic and the cell factors
participate. Most of the hemocoagulation plasma factors are the proenzymes and their
activation occurs due to the limited proteolysis and is accompanied by the peptide
inhibitors cleavage. They are designated the Roman figures. There are 13 such factors
in plasma.
88
 The platelets play an important role in a blood coagulation process. They contain a
lot of (more than 30) different substances which deal with the hemostasis process.
Some of them (according to the various literature scientific sources from 5 to 15) are
called the platelet (thrombocyte) coagulation factors that are designated with the
Arabic numerals.

4. Materials for auditory self-work

4.1. List of study practical tasks necessary to perform at the practical class.
It’s necessary for work: microscope, aggregatograms, Goryaev’s chamber, covering
glasses, plasma rich in platelets, aggregatograph.

Task 1. Bleeding duration determining (by Duke).

To prick the finger on the depth of scarificator point. To absorb flowing blood in
every 30 sec in filter paper. Under norma bleeding duration is 2-4 minutes.

Task 2. Aggregatogram analysis principle.

Diagnostic value: it can give information about thrombocytes qualitative defects and
platelets dysfunctions, platelets quantitative changes at different pathologic processes.
This platelets’ function disturbance will be accompanied by hemorrhages from gums,
oral mucosa.
Main aggregatogram indexes:
1. Aggregation angle (α – L on the figure) – index which reflects aggregation coming
velocity; it is determined by aggregatogram curve ascent steepness after aggregation
inductor addition.
2. Aggregation time (t1) – it is measured from aggregation initiating (beginning) till
point on aggregatogram curve corresponding to maximal aggregation; it
characterizes aggregation degree.
3. Aggregation latent period duration (t) – index which reflects processes not-
registrated on photoelectrocalorymeter (PEC).
4. Desaggregation angle (в) – index which reflects desaggregation process velocity.
5. Aggregation height (h) – index reflecting aggregation degree; it corresponds to
thrombocytic plasma optical density decreasing descent in course of aggregation.

89
FIGURE 12. Aggregatogram.

5. Literature recommended:
1. Lecture course.
2. Mistchenko V.P., Tkachenko E.V. Methodical instructions for dental students
(short lecture course).-Poltava, 2005.-P.41-42.
3. Mistchenko V.P., Tkachenko E.V. Methodical instructions for medical students
(short lecture course).-Poltava, 2005.-P. 71-73.
4. Mistchenko V.P., Tkachenko E.V. Blood system Physiology //Methodical
recommendations to practical classes for students of medical and dental
departments.-Poltava, 2005.-20p.
5. Kapit W., Macey R.I., Meisami E. The Physiology Colouring Book: Harpers
Collins Publishers, 1987.-P. 138.
6.Guyton – Ganong – Chatterjee. Concise Physiology /Ed. By Dr Raja Shahzad Gull:
M.B.B.S., F.C.P.S., King Edward Medical College.-Lahore, 1998 (1st Edition).-
P.197-198.
7. Stuart Ira Fox. Human Physiology.-8th Ed.-McGrawHill, 2004.-P.369-370, 374.
8. Seeley R.R., Stephens T.D., Tate P. Essentials of Anatomy and Physiology.-The
3rd Ed.-McGraw Hill, 1999.-P.293-294, 300.

6. Materials for self-control:

Control questions
1. Thrombocytes, amount, structure.
2. Platelets functions.
3. Vascular-platelet hemostasis mechanism.
4. Microcirculative hemostasis and its significance in dentistry.
5. Thrombocytopoiesis and its regulation.

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 LESSON 36
BLOOD COAGULATION INVESTIGATION

1. The topic studied actuality.

According to World Health Organization, hemostasiopathies place the first position
among the death reasons in adult population.
Salivary glands secret as well as oral cavity different tissues (parodont, salivary
glands, mucosa) possess expressed thromboplastin features. These features are the
mostly expressed in aged people and in the old. Such an activity is a sign of all saliva
types – submandibular, sublingual and parotid one.
Besides thromboplastin, saliva has also other procoagulants. They are analogues of
V, VII, VIII, IX, X, XIII plasmic factors part of which is probably filtrated from blood.
Saliva can not coagulate because of I and II factors absence.
Anticoagulants (antithromboplastins and antithrombins) were also found in saliva.
Their level is less comparatively to the one of procoagulants. They have no such an
importance in physiological and pathological processes in oral cavity.

Physiological bases of measurements at prolonged bleeding after tooth

extraction
They include:
1) careful local haemostasis by means of making tampons with thrombin,
haemostatic spongea and epsilone-aminocapronic acid;
2) in parallel to this – replacement therapy taking into account blood coagulation
factors deficiency.

Physiological basement of patients preparation to tooth extraction at blood

diseases
Such patients can have complications as bleedings after operations. That’s why
doctor before dental manipulation performance must ask the patient whether he had
prolonged bleedings at wounds or operations. If it is necessary the patient must be
consulted in hematologist. In separate patients with blood diseases dental operations
should be done at in-patient department.
At hemophilia in patient dentist should follow systemic approaches 1st stage:
• anamnesis taking;
• X-ray examination;
• special protective plates making from plastic mass (such plates are putted right
before the operation or in the operation day).
The IInd stage – operational one. Local anaesthesia should be done with thin needle
application. All manipulations on tooth extraction are sparing with maximal mucosa
ruptures. They perform hemostasis with haemostatic sponge application, thrombin,
aminocapronic acid and protective plate. They perform replacement therapy by
hemopreparations injecting (plasma +necessary coagulation factors).
The IIIrd stage – post-operational. Replacement therapy continuation.

91
 Complications occurring after tooth extraction in patients with blood
coagulation disorders
In such patients there can be long-termed massive bleedings from oral mucosa at its
traumas and especially after tooth extraction. In patients with haemophilia tooth is
more widely spread. It is delt with teeth mineralization disorder on the background of
total decalcination due to frequent bleedings. Frequent bleedings lead to clots
appearance in cavity and thus to bacterias and inflammation development.
2. Study aims:
To know: processes sequence during coagulational hemostasis, its phases; salivary
glands role in coagulational hemostasis.
To be able to: analyze express-coagulogram.
3. Pre-auditory self-work materials.
3.1.Basic knowledge, skills, experiences, necessary for study the topic:
Subject To know To be able to
Biology and Inheritance main ways Analyze primary coagulopathies
Medical Genetics inheritance ways
Pathophysiology Blood coagulation reasons, Interpretate data received about
classification, blood analysis blood coagulation, coagulation
changes and developmental time
mechanisms
Pediatry with Coagulative hemostasis Say about coagulation in children
Neonatology peculiarities in children of
different age
Internal Diseases Secondary hemostasis Tell about ethiology,
mechanism, its importance in pathogenesis, treatment and
general medicine prevention of secondary
hemostasis disorders
Surgery About ethiology, pathogenesis, To prevent and to liquidate
treatment and prevention of complications after teeth
secondary hemostasis disorders extraction in patients with
in part thromboses and coagulation hemostasis disorders
thrombophilias
Dentistry Oral cavity organs role in To prevent and to liquidate
secondary hemostasis, complications after teeth
complications after teeth extraction in patients with
extraction in patients with secondary hemostasis disorders
coagulation disorders

3.2. Topic content

Blood coagulation is an enzyme process where both the plasmic and the cell factors
participate. Most of the hemocoagulation plasma factors are the proenzymes and their
activation occurs due to the limited proteolysis and is accompanied by the peptide
inhibitors cleavage. They are designated the Roman figures. There are 13 such factors
in plasma.

92
 The platelets play an important role in a blood coagulation process. They contain a
lot of (more than 30) different substances which deal with the hemostasis process.
Some of them (according to the various literature scientific sources from 5 to 15) are
called the platelet (thrombocyte) coagulation factors are designated by the
ArabicCiphras.
In the erythrocytes one can find a number of substances like the platelet ones. They
are known as the erythrocyte blood coagulation factors. They have no figure
designation. The leukocytes have the coagulation factors called leukocyte factors. For
example, monocytes and macrophages upon antigen stimulating synthesize the protein
thromboplastin part namely apoprotein III (tissue factor).
Tissue factors the main component of which is thromboplastin play a significant
role in a blood coagulation. Thromboplastin or tissue factor consists of the protein part
apoprotein III and the phospholipid complex and it is often considered to be a cell
membrane fragment. Upon the tissue destruction or endothelium stimulation by means
of proinflammatory cytokines or endotoxin the tissue factor can be released in a blood
circulation. In various blood circulation regions in the vessels its content differs (e.g.
in veins and arteries, lower or upper extremities, on the right or on the left in ones of
the same name).
Plasma blood coagulation factors
I. fibrinogen – protein, synthesized in liver, transforms into fibrin in course of blood
coagulation. Fibrinogen is also essential for platelet aggregation, tissular reparation.
Normal values into blood – 2-4 g/l (minimal level – 0,8 g/l). One can meet hypo- and
hyperfibrinogenemy.
II. prothrombin – glycoprotein, synthesized in liver at vitamin K presence.
Prothrombin transforms in fibrin under prothrombinase. Norma: 0,1-0,15 g/l. Minimal
level – 40%. One can tell about hypo- and hyperprothrombinemia.
III. thromboplastin – consists of protein apoprothein III and phospholipid complex.
It is in membrane structure of many tissues. It represents matrix for prothrombinase
formation on external way.
IV. calcium – is essential for prothrombinase production, platelet aggregation,
releasing and retraction reactions. Under norma: 0,03-0,04 g/l. Blood coagulation
process remains normal until calcium level reducing to fits development.
V. accelerator-globulin – protein, synthesized in liver, is activated by thrombin, is
in prothrombinase complex composition. Norma – up to 0,01 g/l. Minimal level -10-
15%. Owren’s disease or parahemophily occurs at its absence.
VII. proconvertin – glycoprotein, vit K is essential for its synthesis, is synthesized
in liver. Participates in prothrombinase formation on external way. Norma: about 0,005
g/l, minimal level – 5-10%. Alexander’s disease or parahemophilia occurs at its
absence.
VIII. antihemophilic globulin (AHG) – glycoprotein, it is formed in liver, spleen,
vascular wall. It is essential for prothrombinase formation on internal way. It forms
complex with FW in plasma. Norma: 0,01-0,02 g/l. Minimal level – 30-35%.
Hemophilia A appears at its absence or strong reducing of its concentration.
IX. Cristmas’ factor, antihaemophilic factor B – glycoprotein, is formed in liver at
vit K presence, takes part in prothrombinase formation on internal way. Norma: 0,003
93
g/l. Minimal level- 20-30%. Hemophilia B (Christmas’ disease) is developed at its
absence or strong reducing of its concentration.
X. Stuart-Prawer’s factor - glycoprotein, is formed in liver at vit K presence. It is
prothrombinase complex main part. Norma: 0,01 g/l. Minimal level - 10-20%.
Hemophilia D (Stuart-Prawer’s disease) is developed at its absence or strong reducing
of its concentration.
XI. Rozental’s factor, plasma thromboplastin predecessor - glycoprotein, is formed
in liver, takes part in prothrombinase formation on internal way. Norma: 0,005 g/l.
Hemophilia C (Rozental’s disease) is occurred at its absence or strong reducing of its
concentration.
XII. Hageman’s or contact factor - protein, is activated by negatively charged
surface, adrenaline, kallikrein. It triggers prothrombinase and fibrinolysis external and
internal ways. Norma: 0,03 g/l. Bleeding doesn’t occur even at its concentration
decreasing up to 1 per cent.
XIII. fibrinase, fibrin-stabilizing factor (FSF) – globulin, is synthesized by
fibroblasts, megalocaryocytes; it stabilizes fibrin. It is necessary for reparational
processes normal course. Norma: 0,01-0,2 g/l. Minimal level: 2-5 per cent.
XIV. Fletcher’s factor, prekallikrein- protein, participates in XII-th factor,
plasminogen, high-molecular kininogen activation. Norma: 0,05 g/l. Minimal level -
1%.
XV. Fitzgerald’s-Flozhe factor, high-molecular fibrinogen – is activated by
kallikrein, is involved in XI, XII-th and fibrinolytic agents activation. Norma: 0,06 g/l.
Minimal level: 1 per cent.
The blood coagulation process may be divided into 3 phases.

94
 SCHEME 2. COAGULATION HEMOSTASIS
The first one includes the complex of consequent reactions leading to the
prothrombinase forming. The prothrombinase forming can be realized via two routes:
extrinsic (from injured tissue) or intrinsic (from blood).
The extrinsic route of the prothrombinase forming provides the obligatory presence
of the thromboplastin (or Factor III, tissue factor). The prothrombinase forming via the
extrinsic route begins with the factor VII activation by the interaction with the
thromboplastin. In its turn, the factor VII transforms the factor X into the active state.
Further the factor Xa activates the factor V. The factors III+IV+ Xa +Va form the
complex compound named the prothrombinase. Via the extrinsic route the
prothrombinase is synthesized very quickly (it takes the seconds!).
The factor XII (the contact factor) is an important initiator of the intrinsic
prothrombinase forming route. The kallikrein and high – molecular kininogen (HMK)
are the participants of this reaction. The contact factor is activated by any injured
surface, skin, the collagen, the adrenaline and transforms the factor XI in its active
95
state. The XIa influences directly the factor IX, transforming it into the factor IXa. Its
specific activity is directed to the factor X protheolysis (converting it into its active
form) and occurs on the platelet phospholipid surface at the necessary factor VIII
participating. The whole factor complex on the phospholipid platelet surface received
its name as the thenase ( the thenase complex). As it was mentioned above, the
kallikrein and high – molecular kininogen (HMK) are the participants in a blood
coagulation process by means of which the extrinsic and intrinsic routes combination
takes place. The intrinsic pathway is more prolonged in time (up to 5-6 minutes) as it
is accomplished with a great number of different blood coagulation factors. It is also
implemented without vessel wall injuring (e.g. at the adrenaline concentration
increasing that activates the factor XII).
The second phase of blood coagulation is a transition of prothrombin to thrombin
which is performed by the prothrombinase. It is a proteolytic prothrombin cleavage
resulting in the enzyme thrombin presence. This enzyme possesses the coagulative
activity. It takes only several seconds.
The third phase of blood coagulation is a fibrinogen transition to fibrin. At first
under the influence of the thrombin two fibrin-peptides A and two fibrin-peptides B
are released. As a result of it the fibrin-monomere is formed. Further, the soluble fibrin
is formed due to the polimerization process. But because of the XIII factor (fibrinase)
activation its transition into the insoluble fibrin (fibrin-polymere) is taking place. Next,
this fibrin plug is reinforced thanks to the platelets action (they release the protein
thrombosthenin). This process is known as a retraction. The plug in its turn is named
a clot. The fibrin net becomes gradually tight. That’s why the clot causes the vessel
occlusion and the bleeding is ceased.
One observes II,VII,IX,X,XI,XII and XII factors physiological decreasing in new-
borned. On the contrary, V and VIII-th coagulational factors concentration is at adult
level in them. In low-weighted, immature new-borned one can see more expressed
decreasing of these factors.
Umbilical cord bandaging terms and first child attaching to mother’s breast time
influence greatly on haemostatic indexes. One must hurry up with the first and to
perform the second as soon as possible. On the 3 rd day of child birth procoagulants
level is decreased that leads to hypocoagulation. Further, blood coagulation factors
begin their increasing practically till adult level.
4. Materials for auditory self-work
4.1. List of study practical tasks necessary to perform at the practical class.
Materials and methods: thromboelastograph, saliva, thromboelastogram, water bath,
seconds-meter, plasma, 0,27% solution of CaCl2, 5% solution of CaCl2, capillaries,
filter paper, centrifuge, thromboplastin, plasma.

Task 1. To study thromboelastogram.

Main thromboelastogram’s indexes are the following:
• Reaction time (R) – is measured on direct line from record beginning till
thromboelastogram curves dilation in 1 mm. Given segment corresponds to blood
coagulation invisible phase, i.e. prothrombinase formation. Norma: 9-14 min.

96
• K-segment from R end till thromboelastogram dilation in 20 mm. It is blood
coagulation visible phase, clot formation time; it depends on forming thrombin
concentration and fibrinogen amount. Norma: 5-8 min.
• mA – thromboelastogram branches divergence maximal amplitude is linked mainly
with fibrinogen concentration, thrombocytes amount and functional activity.
It’s necessary to take into account for thromboelastogram analysis that diagram
band velocity is 10 mm per 1 minute. Norma: 48-52 mm.

FIGURE 13. Thromboelastogram.

Task 2. Express-coagulogram
It is laboratory tests set providing preliminarily but quite exactly to determine blood
coagulation and fibrinolysis disorders. Doctor can prescribe differentiated
coagulogram after its assessment in patient on its concrete nosologic form.
Express-coagulogram (normal values):
1. Coagulation time (by Li-White) 4-8 min
2. Thromboelastogram: R 9-14 minutes
K 5-8 minutes
mA 48-52 mm
3. Thrombocytes 180-400 x 109/l
4. Thrombocytic aggregation: spontaneous absent
on ADP present
5. Recalcification time 180-400 sec
6. Prothrombin time by Quick 11-15 sec
7. Thrombin time 12-17 sec
8. Fibrinogen 2-4 g/l
9. Ethanol test negative
10. Prothamine-sulphate test negative
11. Fibrinogen “B” negative
12. Activated partial thromboplastin time (APTP) 25-35 sec (35-50 sec)
97
 13. Fibrin or fibrinogen degrading products less than 0,5 mg/ml or 7,3-3,9
(FDP) or D-dimeres mg%
14. Fibrinolysis (probe on accelerated reaction) 120-240 min (10 min)

Blood coagulation time by Li-White – coagulation time of venous blood in the test-
tube (up to clot formation). This method is not sensitive, it can only tell about general
direction of coagulation (if it is less than 4 min one can tell about hypercoagulation
because blood coagulates faster under such conditions; more than 8 min –
hypocoagulation because blood coagulates slower than under norm).
Thromboelastogram is registered as blood coagulation process objective index.
Platelets amount and their aggregation give us the information about microcirculative
hemostasis.
Recalcification time – general coagulation test which reveals the rudest disorders in
blood coagulation system. The method essence: calcium ions activating blood
coagulation under physiological conditions are neutralized by sodium citrate
corresponding amount. It defines blood liquid state in organism (one mechanism).
Prefix re- means repeated process. They add calcium for clot formation and measure
clotting time. Factors deficiency participating in prothrombinase formation internal
way influence in the biggest extent on test values. If it is more than 140 sec than it can
testify to blood coagulation deep deficiency, thrombocytopeny, heparinization
(anticoagulant heparin taking). This state is named as hypocoagulation. Index value
less than 80 sec (hypercoagulation) is observed at thromboses, disseminated
intravascular syndrome (see below), hyperviscosity, erythrocytosis.
Prothrombin time – this time prolongation at normal fibrinogen content and normal
thrombin time testifies to deficiency of one or some prothrombin complex factors (II,
VII, IX and X). One should think about hypo- or dysfibrinogenemia or anticoagulants
excess (heparin, fibrinolysis products et al.) into blood at simultaneous thrombin time
prolongation.
Thrombin time – characterizes antithrombin, fibrinogen, heparin content. It is
prolonged at antithrombin excess into blood, hypofibrinogenemia, hypoheparinemia,
fibrin and fibrinogen degrading products excess in plasma (these FDP possess
anticoagulant action). These states can be at disseminated intravascular syndrome (see
below), massive thromboembolism. The index prolonging at fibrinogen normal values
takes place at some fibrinogen molecular anomalies. Thrombin time maximal
prolonging is possible at expressed hyperfibrinogenemy as well as at highly-molecular
proteins or paraproteins presence in blood that can absorb procoagulants from plasma
(for instance paraprotein as lg hard chain at hard chains disease, Bens-Jons' protein at
myelomic disease). Plasma complete non-coagulation under thrombin influence is
observed at significant hyperheparinemy and during the third hypocoagulation phase
of hard disseminated intravascular coagulation syndrome.
Activated partial thromboplastin time (APTP) or kaolin-kephalin time represents
highly-standartized coagulative probe sensitive only to plasmic coagulation factors
deficiency especially to the XII, XI, IX and VIII factors as well as to anticoagulants
excess in blood. This time prolonging (hypocoagulation) takes place at anticoagulants
increasing. Also it will be bigger than normal values if there will be deficiency of
98
mentioned procoagulants. According to some data, this test represents one of basic tests
for disseminated intravascular syndrome (see below) determining testifying about
hyper-, normo- and hypocoagulation. APTP is applied at control for performed therapy
effectiveness. The time shortening is observed at DIC-syndrome hypercoagulation
stage as well as some thrombophilies. Positive moments: this test excludes platelet
hemostasis disorders while making sensitive only to coagulation hemostasis disorders.
It reflects blood coagulation intrinsic way disturbances. It is considered to be the most
sensitive coagulation test.
Ethanole, prothamine-sulfate and beta-naphthole tests and probe to fibrinogen “B”
allow to determine “paracoagulation” products – fibrin-monomeric complexes. They
are formed as a result of fibrinogen or fibrin decomposition at fibrinolysis activation.
Positive probes testify to blood disseminated intravascular coagulation (DIC). These
tests receive the name “paracoagulative” because other substances except them do not
coagulate at their adding to fibrinogen and fibrinogen degrading products but they do.
These substances adding to PDP results in euglobulins transfer from zole to gel. The
degree of this sediment is designated from one till four pluses (++++). Mentioned
paracoagulative tests interadd each other and they are better to be applied together.
They can be negative at significant hypofibrinogenemy.
D-dimeres or fibrin (fibrinogen) degrading products (FDP) appear at fibrinolysis
(plasmin action to fibrin or fibrinogen). Test essence: antifibrinogen antibodies binding
with FDP in test-serum with latex loaded with fibrinogen. Level increasing is observed
at DIC-syndrome (at the first hypercoagulation phase), thromboses, thrombophilies
(tendency to thrombosis) treatment.
Probe to accelerated fibrinolysis gives the possibility to evaluate blood (plasma)
lythic features.
Some express-coagulogram tests:
• Plasma recalcification time (it is not estimated in English-speaking countries but is
still assessed in some Ukrainian clinics) – 0,1 ml of plasma + 0,1 ml of physiological
solution, to stay test tube into water bath at 37°C; after 30 sec to add 0,2 ml of
0,277% CaCl2. To determine plasma coagulation time by means of second arrow.
• Thrombin time – 0,1 ml of healthy person plasma + 0,1 ml of physiological solution
and in 60 sec after test tube heating in water bath to add 0,1 ml of thrombin standard
solution. To determine clot time formation time on stop-watch.
• Prothrombin time – 0,1 plasma of a healthy person + (after heating on water bath in
course of 60 sec) 0,2 ml of thromboplastin-calcium mixture and to determine clot
formation time on stop-watch.
• Activated partial thromboplastin time course determining. 0,1 ml of platelet-
weakened plasma (it takes after blood centrifuging at centrifuge velocity during 30
min at 30000 rotations per 1 min) + 0,1 ml of kaolin; to shake the mixture; to put
on watery bath for 2 min; the mixture should be shaken in every 10 min for maximal
contact activation; after 2 min 0,2 ml of kephalin and 0,2 ml of calcium chloride is
added to the mixture.
• Fibrinogen content determining – 0,5 ml of plasma + 0,1 ml of 5% CaCl2. To wait
for solid clot appearance, to carry it to paper filter and to dry till dry-air state, to

99
 weight it on torsion weight, to divide the result received into 2. We receive
fibrinogen concentration in g/l.
• Reaction on ethanol (ethanol test) – 0,4 ml of plasma + 0,15 ml of 50% ethanol
solution, to shake up the test tube and to switch on stop-watch. To determine in 10
min at room temperature whether gel clot occurred in plasma. Probe is considered
positive at clot existence.
• Fibrinogen “B” determining. To add 2 drops of beta-naphthol to 0,5 ml of
investigated plasma. To shake up test tube and to leave it at room temperature for
10 minutes. Stock-taking is performed by the following way: plasma sediment (+),
small granulose flakes falling (++), rude flakes falling (+++), clot formation (++++).

5. Literature recommended:
1. Lecture course.
2. Mistchenko V.P., Tkachenko E.V. Methodical instructions for dental students
(short lecture course).-Poltava, 2005.-P.43-44.
3. Mistchenko V.P., Tkachenko E.V. Methodical instructions for medical students
(short lecture course).-Poltava, 2005.-P. 73-74.
4. Mistchenko V.P., Tkachenko E.V. Blood system Physiology //Methodical
recommendations to practical classes for students of medical and dental
departments.-Poltava, 2005.-20p.
5. Kapit W., Macey R.I., Meisami E. The Physiology Colouring Book: Harpers
Collins Publishers, 1987.-P. 138.
6.Guyton – Ganong – Chatterjee. Concise Physiology /Ed. By Dr Raja Shahzad Gull:
M.B.B.S., F.C.P.S., King Edward Medical College.-Lahore, 1998 (1st Edition).-
P.198-202, 209-210.
7. Stuart Ira Fox. Human Physiology.-8th Ed.-McGrawHill, 2004.-P.374-375.
8. Seeley R.R., Stephens T.D., Tate P. Essentials of Anatomy and Physiology.-The
3rd Ed.-McGraw Hill, 1999.-P.294-295, 300-303.

6. Materials for self-control:

Control questions
1. Coagulational factors.
2. Coagulation mechanism.
3. Fibrin and fibrinogen degradation products and their role in hemostasis.
4. Oral cavity role in coagulational hemostasis support.
5. Positive paracoagulational probes at dental diseases.

100
 LESSON 37
DIFFERENTIATED COAGULOGRAM. DISSEMINATED
INTRAVASCULAR COAGULATION (DIC) SYNDROME

1. The topic studied actuality. In saliva there are many substances influencing on
microcirculative (platelet-vascular) hemostasis, blood coagulation and fibrinolysis.
These substances existence in saliva plays significant physiological role and also is
directly important in pathological processes development in oral cavity and its organs.
These factors role is in following: saliva washing oral mucosa encourages local
hemostasis. It is well-known that wounded surface on oral mucosa occurs every day
during eating and possibility of blood vessels is quite big. But bleedings in oral cavity
are stopped quickly due to active salivary procoagulants and, first of all,
thromboplastin.
At the same time, oral mucosa high regenerative ability during small traumas under
physiological conditions is provided mainly due to fibrinolytic agents in saliva. These
agents help mucosa clearance from fibrin plicas and desquamated epitheliocytes.
During latest 209 years in literature question about tight interconnection and
interrelation between hemostatic processes and other protective blood and tissular
systems (antioxidant, immune, complement, non-specific resistance) is discussed. The
same interrelations between mentioned reactions are also present in oral cavity both in
saliva and in tissular level. In given substrates one can find free radicals, antioxidants,
immunocompetent cells, non-specific defense factors. Their activity and level are
changed in saliva and tissues at some pathological reactions.
For example, at stomatites, parodontitis, jaws fractures and other pathological
processes in oral cavity saliva (and corresponding tissues) stimulating influence on
microcirculative hemostasis state, blood coagulation and fibrinolysis. Saliva
thrombocytoactive and coagulational features enforcement at different-origined
inflammation processes in oral cavity encourages local hemostasis as he result of which
fibrin is formed. Fibrin helps wounded surface repair. But this reaction must not have
excessive character because increased fibrin formation can be unfavorable
phenomenon which disturbs tissue inflamed locus feeding and encourages microflora
growth in this locus (fibrin is a very favorable nutritive environment).
At the same time, fibrinolytic features increasing at this is of essential importance
because it helps tissues clearance from different metabolism products and fibrin
coating. Besides, saliva active fibrinolytic agents can encourage oral cavity tissues
tolerance to hypoxy. Although, excessive fibrinolytic features increasing in saliva can
play also negative role leading to premature fibrin removal (i.e in alveole of extracted
tooth) and thus to slow reparation down. In the first inflammation days when
hyperfibrinolysis takes place in saliva such reaction is essential for wound clearance
from non-alive tissues and products of their decomposition. Then, when wound is clean
and connective tissue granulation has begun excessive fibrinolysis can be unfavorable.
Under such conditions we must inhibit fibrinolysis.

101
 According to literature data, at the biggest amount of pathological processes in oral
cavity are accompanied by hypercoagulation. Such reaction is considered to be the
signal about injure.
At excessive influencings (inflammations, tissues injures at woundings, traumas and
others) in oral cavity one can determine anticoagulant hemostatic link weakening. As
a result, reaction transmits to disseminated intravascular coagulation (DIC).
DIC – is the result of quantitative movements in hemostasis system which lead to
the new state. Main region for all disorders development is microcirculatory vascular
bed. Under one conditions this state has local, under the others – more generalized
character. DIC-syndrome at salivary glands diseases, stomatites, mucosa injuries at
jaws traumas, surgical influencings in this area and other processes development are
more often of local character.
During tissues injure for example salivary glands its decomposition products come
to the blood stream. It becomes key factor of DIC-syndrome development. Such a
reaction in oral cavity has such course like at Artus’ or Sanarelli-Shwarzmann’s
reaction. They are the most typical DIC-syndromes variants in dentistry.
Therapy: just-frozen plasma (blood coagulation factors source)+heparin
(anticoagulants source)+symptomatic therapy.

2. Study aims:
To know: hemostasis disorders at DIC-syndrome, tests set for DIC diagnostics.
To be able to: assess microcirculative and coagulation hemostasis state.

3.Pre-auditory self-work materials.

3.1.Basic knowledge, skills, experiences, necessary for study the topic:

Subject To know To be able to
Biology and Inheritance main ways Analyze primary
Medical thrombocytopathies,
Genetics vasculopathies and
coagulopathies inheritance
ways
Pathophysiology Vascular-platelet hemostasis, Interpret data received about
coagulation reasons, classification, primary and secondary
blood analysis changes and hemostasis and coagulogram
developmental mechanisms; DIC- of DIC-syndrome (at different
syndrome ethiology, pathogenesis, stages) as well; say about
clinics, diagnostics, therapy and primary and secondary
prevention principles; about hemostasis disorders
hemorrhagies main types differential-diagnostical signs
Pediatry with Vascular-platelet, coagulative Treat and prevent primary and
Neonatology hemostasis peculiarities in children secondary hemostasis
of different age; DIC-syndrome in disturbances and DIC-
different-aged children syndrome in children as well

102
Internal Diseases Vascular-platelet, coagulative Treat and prevent primary and
hemostasis peculiarities as well as secondary hemostasis
DIC-syndrome in the adult disturbances and DIC-
syndrome in the adult in
general medicine clinics
Surgery Vascular-platelet, coagulative Treat and prevent primary and
hemostasis peculiarities as well as secondary hemostasis
DIC-syndrome development and disturbances and DIC-
expression in patients with surgical syndrome in the adult in
pathology surgical clinics
Dentistry Coagulogram changes at typical To prevent and to liquidate
pathological processes in maxillar- DIC-syndrome in
facial area; DIC-syndrome stomatological patients
peculiarities in oral cavity

3.2. Topic content

DIC-syndrome is the most dangerous and widely-spread hemostasis pathology type.
Lethality comprises 30-60% in the adults and 70-90% in the new-borned. It is
developed rather more often than it is considered to be present. It is always secondary
as for one or another disease. In turn, DIC can be the reason of many symptoms and
syndromes development. And DIC determines clinical picture of main pathological
process.
DIC-syndrome represents hemostasis system reaction to various pathological agents
influencings. That is why it is not occasional that it has different names in different
countries in part:
• DIC-syndrome;
• “defibrination syndrome”;
• “coagulopathy of consumption”;
• “thrombo-hemorrhagic syndrome”;
• “defibrinative-occlusive syndrome” et al.
But the most suitable is the term “DIC-syndrome”.
Its basis is disseminated blood coagulation in circulation with many microclots and
blood cells aggregates formation. They inhibit circulation in organs and tissues and
cause deep dystrophic changes in them. Hypocoagulation, thrombocytopeny and
hemorrhagy come after intensive coagulation.
This syndrome is universal, non-specific. It is so because it is observed at different
diseases and means catastrophy, accident, similarly to shock. It is accompanied by
blood liquid features loosing and its circulation disturbance in capillaries. Normal vital
activity is impossible under such conditions. Gravity, distribution and velocity of DIC
development vary greatly: from lightened, very rapid, in seconds with lethal result till
latent and durable, chronic; from general blood coagulation till regional and organic
thrombohemorrhagies.
This process can be triggered both with external and internal factors. External ones:
bacterias, viruses, rickettsias, medicines, hypoxy, tissular acidosis and others. Internal

103
ones: tissues decomposition products, thromboplastin appearance in blood, vascular
endothelium injure et al.

Main pathological processes and influences accompanied by DIC-syndrome

development (DIC ethiology)
1. Generalized infections and septic states (bacteriemy, viremy) in part:
• at abortions;
• in deliveries;
• at vessels catheterization;
• at septic shock;
• at infections in the new-borned.
2. All shock types:
• traumatic;
• hemorrhagic;
• burning;
• anaphylactic;
• cardiogenic;
• septic et al.
DIC-syndrome is obligatory and constant shock component that correlates to its
gravity.
3. Traumatic surgical interventions (bleedings, collapse, blood massive transfusions
make DIC-syndrome more frequent).
4. Terminal states:
• predagony;
• agony.
5. Acute intravascular hemolysis and cytolysis (it occurs frequently at
hemotransfusions especially of incompatible blood).
6. Obstetric pathology:
• placenta preliminary self-taking away;
• placenta manual taking away;
• embolism with near-fetal waters;
• fetus intraembryonal death;
• late pregnancy toxicosis;
• Cesar's section;
• abundant hypotonic bleedings;
• womb intensive massage;
• sometimes at physiological delivery.
7. Tumorogenic processes:
• leucosis;
• lungs cancer;
• liver cancer;
• pancreas cancer and the one in other organs.
8. Destructive processes in liver, kidney, pancreas and other organs.

104
 9. Thermal and chemical burnings, esophagus and stomach burnings.
10. Immune and immune complexes diseases:
• systemic red lupus;
• rheumatism;
• rheumatoid arthritis with visceral injuries;
• glomerulonephritis;
• hemorrhagic vasculitis et al.
11. Hemolythic-uremic syndrome.
12. Allergic reactions of medical and other genesis.
13. Abundant bleedings.
14. Thrombocytic thrombocytopenic purpura.
15. Intoxications with snake venoms.
16. Massive hemotransfusions and hemoreinfusions, injections of medicines
containing activated blood coagulation factors.
17. Therapy with medicines causing platelets aggregation, hypercoagulation,
hypofibrinolysis:
• alpha-adrenostimulators;
• synthetic progestins (contraceptive agents!!!!!); that is why oral contraception is
forbidden to women who are predisposed to thrombophyly and moreover possess
varicous disease and thromboses, especially in lower extremities; also women
with the II-nd and the IV-th blood groups are tended to have hypercoagulation as
it has been mentioned below;
• fibrinolysis inhibitors;
18. Wrong applying fibrinolytics and anticoagulants in dosages causing
antithrombin III and fibrinolysis exhaustion.
19. Therapy with defibrinating medicines such as:
• arvin;
• ancrod;
• defibrase;
• reptilase et al.
20. Multiplied and giant angiomes.
Central pathogenesis link – thrombinemia and exhaustion of mechanisms
preventing platelets aggregation and blood coagulation. Thromboplastin is blood
coagulation process trigger. This substance comes into blood stream from damaged
tissues during traumas, operations, necroses, destructions and others as well as with
near-embryonal liquid. Tissular thromboplastin (salivary particularly) plays dominant
role in DIC syndrome development in oral cavity. Thromboplastin can be produced by
damaged endothelium with platelets participation during immune, immune-complex
damages as well as endothelium damage with toxins, hemolysis products et al. Tissular
thromboplastin is synthesized by macrophages (monocytes) and it plays essential role
in DIC-syndrome pathogenesis at bacteriemies, endotoxinemy as well as at immune
and immune complexes diseases. DIC-syndrome at malignant tumors deals with blood
coagulation activating with proteases associated with tumorogenic cells as well as
tissular thromboplastin major mass. Cancerogenic thromboplastin belongs to the

105
strongest among all known thromboplastins. Also monocytes can be thromboplastin
source at many cancer types.
Very important DIC distinguishing feature is other protheolytic systems activation
– fibrinolytic, kallikrein-kinin and complement (so-called “protheolytic burst”).
Antithrombin III (main anticoagulant) level in plasma is diminished during DIC-
syndrome. Similarly, fibrinolytic system components and its activators are consumped.
Thrombinemy leads to blood intravascular coagulation not at once. First, part of
fibrin-monomers appearing under thrombin influence forms soluble complexes (fibrin-
monomeric) or soluble fibrin. This limits blood intravascular coagulation, provides
complexes fibrinolysis. Coagulation appears after 20-24% of fibrinogen coming in
soluble complexes. Thus, fibrinogen is transformed into 2 fibrin types – soluble and
coagulative one. Their correlation determines microcirculation blockage expression as
well as dystrophic changes gravity in kidney, lungs and other organs. That is why
soluble fibrin tests expression (so-called paracoagulation tests) are used for
thrombinemy criterium diagnostics.
Bleedings are determined by disorder both of blood coagulation (anticoagulant
action of fibrin and fibrinogen degrading products, coagulation factors consumption)
as well as vascular-platelet hemostasis – protheolysis products toxic influence on
vascular wall, platelets aggregation and coming out of the blood stream. Both
thrombocytopeny and thrombocytopathy represent hemorrhagies essential factor at
DIC-syndrome.
The syndrome pathogenesis and gravity depend on organs microcirculative
disturbances as well as their dysfunction degree. Complications and death reasons are
as follows as:
• shocked lung;
• acute kidney insufficiency;
• acute circulative insufficiency;
• acute respiratory insufficiency;
• more seldom – liver diseases with parenchymatous jaundice development.
Hemostasis disorders have 4 main stages at DIC-syndrome:
• hypercoagulation;
• different-directed changes (one indexes group testifies to hypercoagulation,
another one - to hypocoagulation);
• hypocoagulation;
• ending (recovery or death).
DIC types:
• lightning;
• acute;
• subacute;
• chronic.
As both hypercoagulation and hypocoagulation are observed, main treatment
measurements comprise:
• fresh-frozen plasma – procoagulants source;
• heparin – anticoagulant.
106
 Preventive physiologic methods: healthy life style:
• constant (regular) physical training (in trained organism anticoagulants and
fibrinolysis activators content is always bigger);
• individual restricted feeding: animal products especially rich in fats will enforce
blood coagulation reactions, plant food – will weaken it;
• not to smoke.

4. Materials for auditory self-work

4.1. List of study practical tasks necessary to perform at the practical class.
Materials and methods: coagulograms sets.

Task 1. Coagulogram for DIC-syndrome (disseminated intravascular

coagulation) diagnostics
Next coagulogram (tests set) is essential for proper DIC-syndrome diagnostics:
Norm:
1. Thromboelastogram: R 9-14 minutes
K 5-8 minutes
mA 48-52 mm
2. Thrombocytes 180-400 x 109/l
3. Thrombocytic aggregation: spontaneous absent
on ADP present
4. Thrombin time 15-18 sec
5. Fibrinogen 2-4 g/l
6. Antithrombin III 80-100%
7. Ethanole test negative
8. Prothamine-sulphate test negative
9. Fibrinogen “B” negative
10. Fibrinogen degradation (derivative) products 7,3±3,9 mg %
11. Euglobuline fibrinolysis 120-240 min

DIC-syndrome is rather widely-spread pathological state. Particularly, such reaction

is observed at:
• shocks different forms;
• sepsis;
• acute intravascular hemolysis;
• massive hemotransfusions syndrome;
• therminal states;
• acute kidney insufficiency;
• malignant tumors;
• traumatical operations;
• oesophageus and stomach chemical burns;
• obstetric pathology;
• physiological labors;
• intoxications and so on.
107
 Possible DIC-syndrom reasons in dentistry:
• after shock;
• facial-mandibular region traumatic injuries;
• malignant tumors;
• facial-mandibular region abscesses and phlegmones;
• jaws fractures and so on.
Acute DIC-syndrome determining is elicitated by the fact that it may be one
hemostasis disorder at some pathology types. For example, at shocks, therminal states,
sepsis hard forms, massive traumas and burns, acute intravascular hemolysis DIC is
disease constant component, its inalienable part. DIC is diagnosed similtaneosely with
main disease recognizing and its therapy is begun immediately.
First, for DIC-diagnosis one can perform simple methods: blood coagulation general
time (norma: 5-8 min), prothrombin and thrombin time, paracoagulational tests indexes
(ethanole, protamine-sulphate), platelet amount. Then one can add also other tests that
prove DIC-syndrome picture.
In a whole, in clinic practice for DIC-syndrome diagnostics one should perform next
tests: platelet amount, platelet aggregation, fibrinogen content, APTT (activated partial
thromboplastin time in plasma pour on thrombocytes), recalcification time,
prothrombin time, antithrombin III (in plasma pour on platelets), fibrinogen
degradation products, soluble fibrin-monomers determining – ethanole and prothamin-
sulphate probes (in plasma pour on thrombocytes), fibrinolysis (in plasma pour on
thrombocytes), fragmented erythrocytes determining.
Changes at acute DIC-syndrome form:
• platelets amount is up to 150 x 109/l and less;
• fibrinogen concentration is up to 1,5 g/l and less;
• activated partial thromboplastin time is up to 50 sec and more (norm: 30-40 sec) –
hypocoagulation sign;
• recalcification time is up to 80 sec and more.
These changes are connected with plasma factors consumption as well as fibrin
derivative products antithrombin action. Other disorders are the following:
• prothrombin time prolonging (due to platelets and blood coagulation factors
consumption as fibrin derivative products antithrombin action) – at acute and
subacute forms;
• thrombin time prolonging on 10 seconds comparatively to normal indexes (it is
linked with hypofibrinogenemia and fibrin derivative products antithrombin
action);
• at platelets aggregation investigation with main biological stimulators (ADP,
adrenaline, collagen) in patients with acute and subacute DIC-syndrome forms
expressed platelets hypoaggregation is observed as a result of
hypothrombocyteaemia and transitory hypofunction caused by thrombine, ADP,
adrenaline, prostaglandine and other proaggregants action to thrombocytes (it leads
to platelets degranulation);
• positive probes on gel-formation with ethanol and prothamine-sulphate are very
essential at DIC-syndrome diagnostics: intermediate products of fibrinogen
108
 transformation in fibrin are occurred in blood in course of this syndrome; these
substances form fibrin-like gel at presence of mentioned substances (ethanol, beta-
naphtol and prothamine-sulfate); such a phenomenon is named as paracoagulation;
under norma concentration of these products is still small that ethanol and
prothamine-sulphate don’t cause gel formation (negative probe); fibrin degradation
products content is more than 10 mcg/ml at DIC-syndrome that is delt with
fibrinolysis secondary activation and plasmin occurrence in blood in concentrations
significantly more than under norma; sometimes it leads to decomposition of not
only fibrinogen and fibrin but also other blood coagulation factors.
For secondary fibrinolysis fast diagnosis one of optimal test is the following: healthy
person native blood, healthy and sick person native blood mixture + thrombin; then to
observe formed blood clots dissolving. At high fibrinolysis level blood clot formed in
healthy and sick blood mixture is dissolved before observant eyes (melt like sugar in a
cup of tea); at the same time blood clot of a healthy person doesn’t dissolve for many
hours.
DIC-syndrome specific expression is microangiopathic hemolytic thrombotic
anemia, signs of which are observed at all this syndrome forms. Its essence is in
following: fibrin fibres are accumulated in microcirculative vessels; these fibres injure
erythrocytes stroma and retard their passage through capillaries. As a result of this
erythrocytes haemolysis acceleration occurs; their osmotic resistance decreasing,
plasma satiation with bilirubin (free, non-conjugated).
Proggressing antithrombin III content decreasing is one of the earliest DIC-
syndrome sign. Being main physiological anticoagulant, antithrombin III reacts to any
activation of haemostatic system procoagulant link. Given progress is the most
expressed in DIC-syndrome patients in consumption coagulopathie stage. It explains
heparin application ineffectiveness without simultaneous antithrombin III
concentration introduction (it contains in fresh-frozen plasma or the “freshest” warm
donor blood but in conserved blood antithrombin III is absent!)
If it’s impossible to perform all mentioned tests under laboratory conditions then
obligatory for DIC-syndrome diagnosis are the following methods: platelet number,
prothrombin and thrombin time, probe with ethanole and prothamine-sulphate,
antithrombin III and fibrinolysis. The other methods may be used as addictive and
proving the diagnosis.
But every doctor must take into account that in every specialized clinics DIC-
syndrome course (and its therapy correspondingly) are strongly differentiated.

Task 2. To assess hematomic hemorrhagia type.

Distinguishing signs for this hemorrhagia type are the following: deep, tensed,
painful hemorrhagias in articular cavities, muscles, subcutaneous fat, in retroperitoneal
space and other places. One can observe also spontaneous nasal, nephral, gastro-
intestinal bleedings. Such hemorrhagias type may be at hemophily, at factor VIII or IX
immune inhibitors occurrence in blood (the most often in women after labour and in
pregnant women with immune diseases), at indirect anticoagulants overdosage and
others.
Bleedings from oral cavity are in dentistry at hemophilia.
109
 Typical coagulogram necessary for this hemorrhagia type determining must include
following tests:
Norm:
1. Factor VIII 70-150%
2. Factor IX 70-150%
3. Prothrombin time 12-15 sec
4. Thrombin time 15-18 sec
5. Fibrinogen 2-4 g/l
6. Fibrinogen degradation products 7,3±3,9 mg%
7. Ethanoletest negative
8. Prothamine-sulphatetest negative
9. Fibrinogen“B” negative

Task 3. To assess microcirculative (petekchio-spotted) haemorrhagia type

Distinguishing features are: capillary bleedings, petekchias on skin, painfulless
ekchimoses, gingival, nasal bleedings, menometrorrhagias, bleedings at
othorhinolaryngologic operations.
This bleeding type is observed at all forms of thrombocytopenias, platelets defects,
fibrinogen hereditary deficiency, II, V and X coagulation factors genetic and acquired
deficiency.
In dentistry any gingival bleedings may serve as suspicion to any of these states.
Coagulogram in such a case must include such probes as:
Norm:
1. Thrombocytes 180-400 x 109/l
2.Thrombocytic aggregation: spontaneous absent
on ADP present
on adrenaline present
3. Platelet adhesiveness 20-50%
4. Factor III activity -
5. Factor 4 activity -
6. Clot retraction 48-64%
7.Bleeding time 2-4 min
8. Prothrombin time 12-15 sec
9. Thrombin time 15-18 sec
10.Fibrinogen 2-4 g/l

Task 4. To assess mixed (microcirculative-haematomic) bleeding type

Distinguishing signs are the following: petekchio-spotted bleedings together with
painful, tensed haematomas in subcutaneous and retroperitoneal fat, abdominal cavity,
visceral organs. Described haemorrhagia type is observed at Willebrand’s disease, VII
and XIII factors deficiency, at complex deficiency of prothrombine complex factors
(II, V, VII, X) and of factor XI which is also delt with liver disorders or vitamin K
intestinal absorbtion disturbances, for example, at mechanical jaundice.
Main sign in dental practice is bleeding from oral cavity, gingival bleedings.
Coagulogram must be the following:
110
 Norm:
I. For Willebrand’s disease exclusion:
1. Thrombocytes 180-400 x 109/l
2. Thrombocytic aggregation: spontaneous absent
on ADP present
on adrenaline present
3. Platelet adhesiveness 20-50%
4. Bleeding time 2-4 min
5. Willebrandt’s factor -
II. For II, V, VII, IX and X factors deficiency exclusion:
1. Prothrombin time 12-15 sec
III. For factor XIII (fibrinase) deficiency exclusion:
1. Fibrinase activity determining 70 sec (100%)

Task 5. To get acquainted to doctor tactics at vasculite-purpure and

microangiomatose bleedings types
1. Vasculite-purpure type - is characterized by hemorrhagies caused by multiple
local inflammatory processes in microvessels of skin, mucosa, inner organs (kidney,
lungs, intestine), the most often of immune genesis. Such bleedings are observed at
hemorrhagic vasculitis of Shenlein-Genoch’s, viral fevers. Localization – gingival,
nasal, uterine, pulmonal, gastro-intestinal bleedings. Doctor’s tactics - to propose to
laboratory to determine tests applied for DIC-syndrome diagnosis because this
haemorrhagia type is a characteristic of DIC hemorrhagic phase and it is its
expression.
2. Microangiomatose type – is characterized by strong, long-termed, repeated
bleedings from nose, oral cavity, kidney, lungs, gastro-intestinal tract. It is observed
at hereditary teleangioectasia different variants. Doctor tactics – blood mustn’t been
taken for analysis! It’s necessary to perform only endoscopic investigation because
vessels are not bleeded out of teleangioectases and all haemostatic probes will be
under norm!

5. Literature recommended:
1. Lecture course.
2. Mistchenko V.P., Tkachenko E.V. Methodical instructions for dental students
(short lecture course).-Poltava, 2005.-P.43-44.
3. Mistchenko V.P., Tkachenko E.V. Methodical instructions for medical students
(short lecture course).-Poltava, 2005.-P. 73-74.
4. Mistchenko V.P., Tkachenko E.V. Blood system Physiology //Methodical
recommendations to practical classes for students of medical and dental
departments.-Poltava, 2005.-20p.
5. Kapit W., Macey R.I., Meisami E. The Physiology Colouring Book: Harpers
Collins Publishers, 1987.-P. 138.
6. Guyton – Ganong – Chatterjee. Concise Physiology /Ed. By Dr Raja Shahzad
Gull: M.B.B.S., F.C.P.S., King Edward Medical College.-Lahore, 1998 (1st
Edition).-P.198-202, 209-210.
111
 7. Stuart Ira Fox. Human Physiology.-8th Ed.-McGrawHill, 2004.-P.374-375.
8. Seeley R.R., Stephens T.D., Tate P. Essentials of Anatomy and Physiology.-The
3rd Ed.-McGraw Hill, 1999.-P.294-295, 300-303.

6. Materials for self-control:

Control questions
1. Coagulational factors.
2. Coagulation mechanism.
3. Fibrin and fibrinogen degradation products and their role in hemostasis.
4. Oral cavity role in coagulational hemostasis support.
5. Positive paracoagulational probes at dental diseases.

LESSON 38
FIBRINOLYSIS AND ANTICOAGULANTS. BLOOD COAGULATION AND
FIBRINOLYSIS REGULATION

1.The topic studied actuality. Anticoagulants and fibrinolysis represents powerful

system in any organism that provides blood support in a liquid state. As it was
mentioned above, oral cavity produces substances influencing on fibrinolysis. Maximal
fibrinolytic activity has mixed saliva, less one – sublingual, minimal – parotid. There
are plasminogen, its pro- and activators as well as inhibitors. There exists point of view
that plasminogen activators role in saliva is in salivary ducts conductance preservation.
Any inflammation in its second phase is accompanied by fibrinolytic system
activation.
2. Study aims:
To know: fibrinolysis process algorhythm and the process regulation, main primary
and secondary anticoagulants; blood coagulation and fibrinolysis main regulative
mechanisms.
To be able to: determine blood fibrinolytic activity.

3. Pre-auditory self-work materials.

3.1.Basic knowledge, skills, experiences, necessary for study the topic:

Subject To know To be able to

Pathophysiology Main anticoagulants, their action Explain main
mechanisms; fibrinolysis system pathophysiological
work mechanisms of
anticoagulative and
fibrinolytic systems
disturbances

112
 Pediatry with Anticoagulative and fibrinolytic
Neonatology systems peculiarities in different-
aged children; mentioned systems
disturbances ethiology,
pathogenesis, diagnostics, clinics,
therapy and prophylaxy principles
Internal Diseases Main anticoagulants, their action
mechanisms; fibrinolysis system
work; mentioned systems
disturbances ethiology,
pathogenesis, diagnostics, clinics,
therapy and prophylaxy principles
Surgery Main anticoagulants, their action
mechanisms; fibrinolysis system
work; mentioned systems
disturbances ethiology,
pathogenesis, diagnostics, clinics,
therapy and prophylaxy principles
in surgery
Dentistry Coagulogram changes at typical
pathological processes in maxillar-
facial area; maxillar-facial area role
in anticoagulation and fibrinolysis
Treat and prevent
anticoagulative and
fibrinolytic systems disorders
in different-aged children
Treat and prevent
anticoagulative and
fibrinolytic systems disorders
in internal diseases clinics
Treat and prevent
anticoagulative and
fibrinolytic systems disorders
in the adult in surgical clinics
To prevent and to liquidate
anticoagulative and
fibrinolytic systems disorders
in stomatological patients

3.2. Topic content

Inspite of circulation there are all necessary factors for the clot forming. Under
physiological conditions in presence of uninjured vessels a blood remains fluid. It’s
determined by the presence of components, preventing the blood coagulation
(anticoagulants) in the circulation. Besides, a blood is kept fluid because of the
haemostatic system fibrinolytic components in it.
But it’s necessary to underline that blood doesn’t coagulate in vessels due to others
reasons too. Factors providing this feature are:
• blood stream velocity: it is well-known that where circulation velocity is the less,
the more threat to intravascular blood coagulation (for example, blood is more often
condensed in veins comparatively to arteries and phlebothrombosis,
thrombophebitis is occured); it also observed in blood circulation regions where
circulation is changed, for instance at bifurcations places;
• similar charge (negative) of vascular vessels internal layer;
• formed elements negative charge;
• most coagulational factors negative charge: charge similarity of blood vessels
internal layer and blood coagulation factors creates forces for pushing away; at
vascular walls injuries vascular wall charge is decreased or even is changed onto
positive that creates additional conditions for intravascular coagulation initiating;

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• blood coagulation factors are inactive in blood; blood coagulation are not triggered
until factors are under unactive state;
• there are inhibitors to active blood coagulation factors (VIII a, IXa, Xa, XIIa): even in
a case of any factors activation further process development is not obligatory.
Probably, you paid your attention that blood coagulation is origined from the XIIth
factor activation. It is activated by side surface or hyperadrenalinemia and it becomes
XIIa only after this. Reaction cascade (chain) is begun directed to other blood
coagulation factors activation. Moreover, this reaction is not spontaneous, it looks like
stairs (fall) when one factor activates other one in definite order, in definite sequence.
But anticoagulants are essential factors preventing possible blood coagulation
activating.

The natural anticoagulants are divided into primary and secondary ones. The
primary anticoagulants are such substances that are constantly present in the
circulation. They may be of three groups: antithromboplastines, antithrombines and
fibrin forming inhibitors. Otherwise, all these anticoagulants are the substances that act
depending on the blood coagulation process stage.
The substances preventing the prothrombinase forming are the antithromboplasties
(they are secreted by the vessel wall endothelium, their content in veins is larger than
in arteries), vitamin K-dependent protein C (inhibits the factors V, VIII), protein S, the
endothelium protein – thrombomodulin, the placenta anticoagulant protein and others.
The substances inhibiting thrombin action are antithrombines.They are of different
groups but the most important of them are: antithrombin III and heparin. Antithrombin
III – is a prothein of a globulin origin that is formed in liver, kidneys, spleen, lungs and
blood vessels as well. Its content reduces with the age, its concentration is less in
women as compared with men (NB! Women have the thrombophlebitis and
phlebothromboses more often than men), its content in the pregnant gets smaller. Its
content is smaller in human beings with the II(A) blood group and the people eating
fat food (particularly of animal origin). Its activity decreases at the diseases of those
organs where it is formed. Antithrombin III is a heparin co-factor. Besides, it inhibits
up to 70 per cent of thrombin occuring in blood as well as the factors IXa, Xa, XIa,
XIIa. There are cases of its hereditary insufficiency.
Heparin – is also an antithrombin.It is a polysaccharide transforming antithrombin
III in anticoagulant of immediate action thus increasing its activity. In absence of
antithrombin III heparin possesses a weak anticoagulant activity. Moreover, heparin
without antithrombin III doesn’t prevent the external prothrombinase forming way. So,
heparin efffect may be very weak as a result of antithrombin level decreasing in
patients’ blood that it’s necessary to take into account at its administration. Heparin
also forms the complex combinations with thrombogenic protheins and hormones
which finally possess anticoagulant and fibrinolytic features. Heparin influences the
thrombocyte aggregation, has antiviral action and antiinflammatory properties as well.
In blood heparin can be found in basophiles, in vessels – in mast cells. It is degenerated
by the heparinase enzyme in liver.
Secondary anticoagulants – are the “worked-off” blood coagulation factors (that
participated in blood coagulation process) and degradation fibrin and fibrinogen
114
products or derivates (PDF) having antiaggregative and anticoagulative action. The
secondary anticoagulants role comes to limiting of intravascular blood coagulation and
thrombus dissemination via vessels.
At various diseases there may appear the pathological anticoagulants dealing with
different immunoglobuline classes and inactivating separate blood coagulation factors.
These and some other data are collected in the table.

Table 5. Main primary physiological anticoagulants

Anticoagulant name Action mechanism

Antithrombin III (AT III) Proggressively acting inhibitor of thrombin, Xa factor and,
in less extent, other blood coagulation factors. Heparin
plasmic co-factor.
Heparin Sulphatated polysaccharide forming complexes with AT
III and transforming the latest one into fast-acting
anticoagulant.
Co-factor of heparin II Weak anticoagulant the action of which is expressed at
heparin presence after AT III removal from plasma
Protein C Vit K-dependent serine-amidase inactivating VIIIa and
Va; plasminogen endogenous activator. It is activated by
thrombin and complex “thrombomodulin-thrombin”
Protein S Protein C vit K-dependent co-factor
Thrombomodulin Glycoprotein fixated on endothelium cytoplasmic
membrane. It binds and inactivates thrombin but does not
inhibit its activating action to protein C
Tissular coagulation way Inhibitor of complex “tissular factor-factor VIIa-factor
inhibitor (TCWI or TFPI) Xa-Ca++”
“Contact inhibitors” They disturb coagulation internal mechanism activation
(phospholipid, placentary) (complexes of XII and XI factors)
Antithromboplastines They are inhibitors of complex III-VIIa. It is weak
α2-macroglobuline inhibitor of thrombin, plasmin and kallikrein
α1-antitrypsine I Thrombin, IXa, XIa, XIIa and plasmin inhibitor
complement I inhibitor The same
(Anti-C1)
Fibrin-monomeres They inhibit fibrin formation
polymerization inhibitors

Fibrinolysis – is an integral part of haemostasis system. It always accompanies the

process of blood coagulation and even is activated by the same factors (XIIa, kallikrein,
HМК and others). Being the important defence reaction it prevents the occlusion of
blood vessels by fibrin clots and leads to the vessel recanalization after the bleeding
stoppage. The fibrinolysis components play key role in extracellular matrix removal.
Besides, they regulate the growth and the division of cells, the reparation of wounds,
the regeneration of muscles, the growth and metastasis of tumors etc.

115
 The main enzyme destroying the fibrin is plasmin (sometimes it is called
fibrinolysin), that in a circulation is in non-active state as proenzyme plasminogen.
Under the influence of the activators there occurs plasminogen peptide junctions
cleavage that leads to in it’s turn to plasmin forming. Plasminogen may be found not
only in plasma and in serum but in other types of liquids (sperm, follicules, saliva), in
tissues and leukocytes either. This is a prothein of a globulin origin the biosynthesis of
which is performed in a bone-marrow.
To transform into plasmine plasminogen needs to be activated. Plasminogen
activators are contained first of all in tissues (vessel wall). Tissue plasminogen
activator (TPA) – is mainly formed in vessel wall endothelium. Urokinase as
plasminogen activator is produced in kidneys (juxtaglomerular apparatus), in
fibroblastes, epitheliocytes, pneumocytes, placenta, endotheliocytes either. There are
also plasminogen activators in erythrocytes, thrombocytes and leukocytes.
Except plasminogen activators there exist the fibrinolysis inhibitors in plasma.
Nowadays one can tell about 4 types of plasminogen and urokinase tissular
activators inhibitors.
1) The most important among them is inhibitor of the first type (ITAP-1), which is
often designated as endothelial. Besides, it is synthesized not only by hepatocytes but
also by monocytes, macrophages, fibroblasts and myocytes.
Up to 90 percent of antifibrinolytic activity is contained in platelets alpha-granules
which are released in blood stream at their activation. While accumulation in
endothelium injured locuses platelets release ITAP-1. This reaction has an essential
importance for injured vascular wall restoration.
Fibrinolytic blood activity is greatly determined by the correlation between the
fibrinolysis activators and inhibitors.
2) α2-antiplasmine influencing not only on plasmine but also on urokinase;
3) α1 –protease inhibitor (б1-antitrypsine) – strong plasmine inhibitor;
4) α2-macroglobulin;
5) plasminogee activator inhibitors secreted by endothelium, macrophages,
monocytes and fibroblasts.
Fibrinolysis like the blood coagulation process is performed in three phases.

FIBRINOLYSIS SCHEME:

Internal way External way

(epinephrine and

116
 norepinephrine level
increasing in blood) Vessels injury

Plasminogen
XII XII

Plasminogen tis-
Prekallikrein kallikrein+HMK
sular activator
Hageman-dependent
(PTA)
Urokinase
The others
Plasminogen activators
from erythrocytes
Plasminogen activators from
(erythrokinase),
platelets, erythrocytes, leucocytes
leucocytes, platelets
Hageman-independent

Plasmin

Fibrinogen fibrin

Fibrinogen/fibrin degradation products:

early (A, B, C, X, Y) and late (D, E).

SCHEME 3. Fibrinolysis cascade.

The first phase, the forming and secreting of plasminogen activators may occur in
extrinsic and intrinsic ways. The extrinsic way of plasminogen activation is due to
the TAP, urokinase and some others. The intrinsic way of plasminogen activation is
divided into Hageman-dependent and Hageman-independent. The first of them takes
place under the influence of the XIIa, kallikrein and HMK factors that transform
plasminogen into plasmin. Hageman-dependent fibrinolysis is accomplished very fast
and bares urgent character. Its main designation comes to the circulation clearence
from fibrin clots forming in course of disseminated intravascular blood coagulation
process. The second one can be realized under the influence of proteins “C” and “S”.
In the second fibrinolysis stage under the action of the activators mentioned above
plasminogen transforms into plasmin. Finally, in the third stage, plasmin effects on
fibrin. As a result at first the early (high-molecular) and then the late (low-molecular)
fibrin degradation products or derivates (FDP) appear. The early PDF influence on
the platelet aggregation and blood coagulation thus increasing them. The late PDF are
characterized by the anticoagulant features and effort the fibrinolysis reaction.

117
 Natural ancoagulants and fibrinolytic components level is decreased in new-borns.
In low-weighted, immature babies - more expressed anticoagulants decreasing.
Fibrinolytic components level is reduced on the 3rd day of life that leads to fibrin clot
dissolving time increasing. Further, natural anticoagulants concentration begins its
gradual increasing and becomes normal up to 14th day. Blood fibrinolytic activity
reaches its normal value to this time too. But at the same time, antithrombin III
concentration is remained comparatively low in a child of the 1st month of life.
Vascular-platelet hemostasis, blood coagulation and fibrinolysis regulation.
There exist 4 levels of haemostatic system regulation.
Molecular level – supposes haemostatic equilibrium supporting for factors
influencing on vascular-platelet haemostasis, blood coagulation and fibrinolysis.
Factor excess appearing in organism due to one or other reason must be liquidated in
short time as soon as possible. Such equillibrium is constantly supported between
prostacycline and thromboxanes, procoagulants and anticoagulants, plasminogen
activators and inhibitors. Cellular receptors existence to many blood coagulation
factors underlies haemostatic equilibrium in haemostatic system at molecular level.
Receptors to coagulation and fibrinolysis factors coming off cells (“swimming”
receptors) acquire new features becoming natural anticoagulants, plasmin inhibitors
and plasminogen activator. Regulational molecular level may be realized with immune
system by means of antibodies to activated coagulation and fibrinolysis factors – IIa,
Xa, tissular palsminogen activator and others- formation. There is genetic control under
factors production providing blood clot forming and dissolving.
Cellular level. In circulation constant coagulational and fibrinolytic factors
consumption occurs that must obviousely lead to their concentration restoration. This
process must be caused by either activated factors or their metabolic products. If it is
really so, cells must have receptors to indicated substances. Such receptors were found
on many cells to thrombin, kallikrein, plasminogen activator, plasmin, FDP and others.
Cellular level is also provided by “near-wall” fibrinolysis occurring at fibrin
accumulation on vascular wall endothelium.
Organic level - determine haemostatic system optimal existential conditions in
circulation different regions. Vascular-platelet and coagulational hemostasis and
fibrinolysis mosaic is expressed due to this level. Our chair collaborators scientifical
works for last years have proved that blood while passing through one or other organs
(for example, brain, extremities muscles, kidneys) is satiated with additional
hemocoagulational and fibrinolytic factors which may be synthesized in these organs.
Moreover, we (V.P.Mischenko, I.V.Mischenko, E.V.Tkachenko, E.A.Tkach,
O.V.Kokovskaya, J.M.Grishko and students of different departments and courses
which are members of our chair student’s scientific society) demonstrate that blood
outflowing from these organs on the right and on the left has different coagulative and
fibrinolytic features. It was the base to consider that in animal and human organism
there is haemostatic and fibrinolytic process asymmetry. Such an asymmetry was found
by us in different laboratory animals (hens, rats, rabbits, guinea pigs, cats) and human
beings.

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 Nervous-humoral regulation controls hemostasis state from molecular till organic
level, providing reactions integrity at organism level. It is realized mainly through
vegetative nervous system sympathetic and parasympathetic parts.
First of all, one should mention that there exists cortical (conditioned-reflectory)
hemostatic system regulation. There some scientific data indicating on the possibility
to determine conditioned reflexes both to the acceleration and especially to blood
coagulation retardation up to bleedings (bleeded tears, hemorrhagias in places
analogous to wounds places, caused at Christ crucifixion et al.).
CNS separate structures (cerebellum, thalamus, hypothalamus) participate in
regulation both of activation and inhibition of haemostasis system functionning. As it
was proved (B.I.Kuznic, V.P.Mischenko, L.L.Goncharenko., D.S.Zazykina)
hypersympathicotony (acute hemorrhagia, hypoxy, stress, intensive muscular activity,
adrenalin and noradrenalin introduction) causes blood coagulation acceleration and
fibrinolysis enforcement. It is linked not only with Hageman’s factor activation but
also with thromboplastin, tissular plasminogen activator releasing from vascular wall.
But the most interesting is the fact that at hyperparasympathicotony (vagus irritation,
acetylcholine and pilocarpin introduction) we observe coagulation acceleration and
fibrinolysis too. Under this conditions thromboplastin and tissular plasminogen
activator releasing from vascular wall occurs too. Moreover, drugs vasoconstrictors
and vasodilatators by their nature cause similar answer from blood coagulation and
fibrinolysis - thromboplastin and tissular plasminogen activator releasing. It testifies
that vascular wall is blood coagulation and fibrinolysis efferent regulator!
Hemostasis regulation humoral mechanism - is hormones, mediators, vitamins and
other substances action.
Hormones of suprarenal glands (corticosteroids, adrenalin), hypophysis (ACTH,
STH), thyroid (thyroxine), parathyroid (parathormone) and other glands mainly
activate blood coagulation, although everything depends on their dosage.
Mediators – noradrenalin, acetylcholine and others mainly activate blood
coagulation too.
Vitamins have different influence on hemostasis process.
Vitamin “A” - inhibits coagulation and activates fibrinolysis.
Vitamin “E” – accelerates blood coagulation and suppresses fibrinolysis.
Vitamin “PP” (nicotinic acid) – accelerates coagulation and increases fibrinolysis.
Vitamin “B12” – accelerates coagulation and suppresses fibrinolysis.
Vitamin “C’ - enforces blood coagulation.
But at the same time doctor should remember that this vitamins effect on
hemocoagulation depends on their dosage. These data are quite important because
vitamins and hormones usage is widely-spread.
Hypercoagulation occurs mainly due to time shortening mainly of the first
haemocoagulation stage. That’s why hypercoagulation reasons are quite different and
depend on many coagulation factors located in plasma, formed elements and tissues.
Hypercoagulation reasons:
1) Hypercoagulation occurs at blood coagulation factors (especially I, VIII and IX)
excessment). It may be observed at:
• muscular activity;
119
 • emotions;
• pain;
• hyperadrenalinaemia;
• in pregnant women.
1) Thrombocytosis.
2) Erythrocytosis.
3) Erythrocytic haemolysis:
• burns;
• haemolytic states;
• toxic animals bites;
• blood hemotransfusions.
4) Some leukosis forms.
5) Any tissular injuries.
But hypercoagulation may be transformed into hypocoagulation, which is
secondary under natural conditions and is caused by thrombocytes and plasma
coagulation factors consumption as well as secondary anticoagulants formation.
Primary hypocoagulation reasons are following:2
1) blood coagulation congenital disorders:
• hemophilia;
• thrombocytopathies (Glanzman’s disease et al.);
2) autoimmune hypocoagulation accompanied by bleedings;
3) DIC-syndrome.

Materials and methods: watery bath, stop-watch, centrifuge, plasma, 1% solution of

acetic acid, borate solution, fibrinolysin (plasmin) solution, 0,277% solution of calcium
chloride, distillate water, saliva.

Task 1. Blood fibrinolytic activity determining.

8,5 ml of distillate water + 0,15 ml of 1% solution of acetic acid and 0,5 ml of
investigated plasma, mix and put in a fridge for 30 min. After this test tube is
centrifugated at 1500 rotations per minute in course of 5 min. Then liquid is poured,
test tube is turned over onto filter paper for several minutes to be dry. Investigator must
add 0,5 ml of boric acid to sediment and to dissolve the sediment with glass stick, then
it’s necessary to add 0,1 ml of fibrinolysin solution and 0,1 ml of 0,277% of CaCl 2,
content is mixed and is putted into bath at 37°C switching on the stop-watch. Norma:
120-240 min.
If the index is more than 240 min than it testifies to fibrinolytic activity inhibiting.
If it is less than 130 min – it fibrinolysis activation sign.

Task 2. Fibrinolytic bleeding laboratory diagnostics principles.

Blood fibrinolytic activity increasing as a rule is observed in a case of blood
coagulation activating. That’s why fibrinolysis as hemorrhagias primary reason is a

120
very seldom phenomenon. But in a case of fibrinolysis expressed activation one can
see complete degradation not only of fibrinogen but also other coagulational factors.
Under these conditions real bleeding is developed which is only may be situated among
fibrinolytic bleedings. Unfortunately, doctor in clinical practice without sufficient
bases (without this process laboratory diagnostics) makes the diagnosis “fibrinolytic
bleeding”. Such a situation may be with dentist too at alveolar bleedings after tooth
extraction or other operations in oral cavity. To deny any suspicion (or to prove it, on
the contrary) about possible fibrinolytic bleeding one must send to the laboratory
following tests sets:
Norm:
1. Natural clot lysis 10-20%
2. Probe on accelerated fibrinolysis 10 min
3. Euglobulins lysis time 120-240 min
4. Fibrinogen derivative products 7,3± 3,9mg%
5. Ethanol test negative
6. Prothamine-sulphate test negative
7. Fibrinogen “B” negative

If natural clot lysis index is decreased but probe to accelerated fibrinolysis is

increased with simultaneous fibrinogen derivative products reducing that it testifies to
blood lytic features decreasing. In course of contrary change and positive
paracoagulation probes existence – it is concluded about fibrinolytic system activation.

Task 3. Getting acquaintance with some tests characterizing hemostasis

anticoagulant link
1) Protein C. Normal value is near 1 mg/l or 70-130%.
2) Protein S. Normal value is 60-140%.
3) Antithrombin III (by Byshevsky) – 84-116% or 210-300 mg/l. Antithrombin III
level is reduced at: DIC-syndrome (hypercoagulation phase), thromboses and
thromboembolism.
4) Antiphospholipid antibodies – are absent under physiological conditions.
Antiphospholipid syndrome is observed at various diseases. Antiphospholipid
antibodies recognize phospholipids complexes with plasma proteins participating in
hemostasis process (prothrombin, proteins C and S, thrombomodulin, antithrombin III
and others). Anticoagulants binding decreases organism antithrombotic potential and
increases risk of thrombophilies. Autoantibodies belong to lg G, A, M.
Main antiphospholipid antibodies are:
• anticardiolipin (the mostly often) ;
• antiphosphatidylserin;
• antiphosphatidylinositol;
• antiphosphatidylethanolamin;
• antiphosphatidylcholine.
Autoimmune diseases such as systemic red lupus, rheumatism, dermatomyositis and
some others as well as pregnancy (at varicous disease, accompanying thrombosis in

121
woman) can be complicated by antiphospholipid syndrome development.
Antiphospholipid syndrome can lead to miscarriages. Myocardial infarction also can
carry this nature.

5. Literature recommended:
1. Lecture course.
2. Mistchenko V.P., Tkachenko E.V. Methodical instructions for dental students
(short lecture course).-Poltava, 2005.-P.44-47.
3. Mistchenko V.P., Tkachenko E.V. Methodical instructions for medical students
(short lecture course).-Poltava, 2005.-P. 75-80.
4. Mistchenko V.P., Tkachenko E.V. Blood system Physiology //Methodical
recommendations to practical classes for students of medical and dental
departments.-Poltava, 2005.-20p.
5. Stuart Ira Fox. Human Physiology.-8th Ed.-McGrawHill, 2004.-P.375-377.
6. Seeley R.R., Stephens T.D., Tate P. Essentials of Anatomy and Physiology.-The
3rd Ed.-McGraw Hill, 1999.-P.295.

6. Materials for self-control:

Control questions
1. Fibrinolytic system factors.
2. Plasminogen external and internal activators.
3. Fibrinolysis scheme.
4. Fibrinolysis assessment methods.
5. Oral cavity role in fibrinolysis process.

LESSON 39
TOTAL BLOOD
1. The topic studied actuality. Blood analyze is non-specific, universal, still
judiciousely used diagnostic method. It gives significant information at different
physiological and pathological conditions.
2. Study aims:
To know: physiological limits of main blood indexes; major age peculiarities.
To be able to: interpret total blood (to see indexes abnorm, to say what conditions it
can testify to).
3. Pre-auditory self-work materials.
3.1.Basic knowledge, skills, experiences, necessary for study the topic:
Subject To know To be able to
Pathophysiology Main blood indexes norm Interpret total blood and to tell
about main mechanisms and
probable reasons of the changes
observed

122
 Pediatry with Blood peculiarities in different- Interpret total blood changes
Neonatology aged children in part about the
first and the second crossings
Internal Diseases Total blood main indexes norm To tell about probable reasons of
the changes observed in internal
diseases clinics
Surgery Total blood main indexes norm To tell about probable reasons of
the changes observed in surgical
clinics
Dentistry Total blood main indexes norm To tell about probable reasons of
the changes observed in
stomatological patients

3.2. Topic content

Color index characterizes erythrocytes satiation degree with haemoglobin. It is
calculated on formula:

C.I.= (X haemoglob. x 5,0 x 1012/l) : (167 g/l x X erythroc.)

where:
X haemoglob. – found haemoglobin amount (g/l);
X erythroc.- found erythrocytes amount in 1 l of blood.
The second formula: Hb (g/l) x 3 : RBC (3 first ziphras). It is evaluated in conditional
units.
At English-speaking countries all these indexes are automatically determined
practically in every clinic. Especially they are of great importance for anemias
differentiated diagnostics.
1. MCV (Mean Corpuscular Volume) – average erythrocytic volume.
MCV=HCT (%) : RBC (x 1012/l) x 10, where: HCT- haematocrit; RBC- erythrocytic
amount.

MCV (normocytes) - adults: 78-94 mcm3 or fl (femptolitres)

new-borns: 95-105 mcm3;
children: 76-90 mcm3.
MCV↑ (macrocytosis):
• pregnancy;
• megaloblastic anaemia;
• myelodysplastic syndrome;
• liver diseases;
• hypothyreoidism;
• alcoholism;
• treatment with estrogens;
• treatment with barbiturates et al.

123
 MCV↓ (microcytosis):
1) anaemias:
• hereditary microspherocytic;
• iron-deficient;
• sideroblastic;
• chronic anaemias;
• thalassaemia (hereditary haemoglobinopathy);
2) hypohydration;
3) aluminium intoxication.

2. MCH (Mean Corpuscular Haemoglobine) – haemoglobine average content in

erythrocytes.
MCH=Hb (g/l):RBC (x 1012/l)
MCH (erythrocytic normochromy)- adults: 27-33 pg (picogram)
children: 24-30 pg
MCH↑(hyperchromy):
• new-borns;
• megaloblastic anaemia;
• liver cirrhosis.
MCH↓ (hypochromy):
• iron-deficient anaemia;
• thalassaemia;
• sideroblastic anaemia.

3. MCHC – Mean Corpuscular Haemoglobine Concentration – Mean

haemoglobine concentration in erythrocyte – Hb (g/decaliter): Ht or HCT (l/l) x 100
MCHC (norma): 32-36 g/dl (320-360 g/l).
MCHC↑:
• new-borns;
• hereditary spherocytosis;
• long-termed hypohydration.
MCHC↓ (absolute hypochromy):
• iron-deficient anaemia;
• thalassaemia;
• sideroblastic anaemia;
• hydraemia.

4. Reticulocytes amount in blood volume unit - Norm:

adults and children: 0,2-2,0 % or 25-85 x 109/l;
new-borns : 2-6% or 85-250 x 109/l.
Reticulocytosis (increasing):
• anaemias (haemolytic, acute posthaemorrhagic),
• in initial period (6-10th days) of effective anaemias treatment, caused by iron and
folic acid, cyancobalamine and pyridoxine insufficiency;
124
• in course of exit from bone marrow hypoplasy after therapy with cytostatics;
• after splenectomy;
• at malaria.
Reticulopenia (decreasing):
• hypo- and aplastic anaemias;
• megaloblastic anaemias;
• acute leukemias;
• radiation disease;
• in course of cytostatic therapy;
• pre-regenerative crisises at haemolytic anaemias;
• kidney diseases;
• radiation disease anaemia.

5. Reticulocytic index (RI)= R (%) x Ht (of patient) : Ht (normal). It is used for

more adequate bone marrow erythropoietic activity assessment with the haematocrit
taking into account.
Norm: 1%
RI ↑:
• haematocrit decreasing;
• haemolytic anaemias (due to erythropoiesis activation);
• initial stage of effective anaemias treatment (due to the same reason).
6. Reticulocytes formation index – RFI=RI:t (reticulocytes maturation time in
perypheral blood) x 10.
RFI (norm)=1 cond. un.
RFI (at anaemia)>3 indicates to erythropoietic cells prolipheration and maturation
activating.
RFI (at anaemia)<3 indicates to erythropoiesis inhibition.

7. RDW – erythrocytes distribution dispersion by volume – standard inclination

correlation to MCV.
It is estimated by erythrocytometric curve (of Price-Jons’) variation co-efficient and
is expressed in percentage.
Anisocytosis (this index increasing) – different-sized Er presence in one blood
smear. It is characteristic for anemias (hemolytic, Fe-deficient, megaloblastic) as well
as osteomyelofibrosis.

Table 6. Erythrocytes distribution by their size

Term Er average diameter (mcm) MCV (fl)
Normocytic 6,8-8,5 78-94
Microcytic <6,8 <75
Macrocytic >8,5 >94

OTHER HEMATOLOGIC INDEXES WIDELY USED IN MODERN CLINICS

AND LABORATORIES
125
• NRBC/100 WBC - Er amount on 100 L;
• TOXIC GRAN – irritation granules (appearing in blood at intoxications);
• Dohle Body – specific granules (Dohle bodies);
• RPI – Reticulocytic-Platelet Index;
• Aniso – anisocytosis ;
• Мacro – macrocytosis;
• Micro – microcytosis;
• Poikilo - poikylocytosis;
• Ovalocyte – ovalocytosis;
• Elliptocyte – elliptocytosis;
• Target cells – targeted Er;
• Shistocyte – shictocytes (“hedgehogs”);
• Acanthocyte – “hedgehogs”, but “needles” amount is less, they are thicker and
located more seldom;
• Tear drop – dacryocytes (Er like tears);
• Spherocyte;
• Sickle Cell – drepanocyte, sickle-shaped Er (it contains Hb S, badly attaching and
giving oxygen, it possesses increased ability to sedimentation and vessels
obstruction and is observed at sickle-celled anemia;
• Hypochromia;
• Polychrom – polychromatophilia – sensitivity to many stains;
• Howel-Jolly (bodies) - (leucocytes granules);
• Burr Cell or bodies – sexual chromatin (is detected in neutrophils).
TOTAL BLOOD EXAMPLE
40-YEARED MAN
(it is performed on automatized counter MS9)
• WBC 6180 /ul (norm 4000-11000) or 6,18 x 109/l
• RBC 4,57 Mil/ul (norm 4,20-5,40) or x 1012/l
• RDW 11,2
• Hemoglobin 9,5 g/dl (norm 12-16)
• Hematocrit 32,1 % (norm 36-46)
• MCV 70,2 fl (norm 80-100)
• MCH 20,8 pg (norm 27-32)
• MCHC 29,6% (norm 33-38)
• ESR 8 ihr (mm/hr) (norm up to 15)
• ANISO 2+
• MICRO +
• MACRO +
• HYPOCHROMIA +
• POIKILO –
• Neutrophils 72%
• Lymphocyte 27%

126
• Monocyte 1%

Result: Anemia Hypochromic Microcavitary

Table 7: Main indexes of blood:

RBC Red Blood М 4,5-5,5х 1012/l Newborns After 75 years –
Cells W 3,7-4,5 х 1012/l 4,0-7,0 х 1012/l physiological
babies, children anemia
3,7-5,3 х 1012/l
HB Hemoglobin М 130-180 g/l Newborns 200- After 75 years –
W 120-160 g/l 240 g/l; physiological
babies 110-140 hypochromy
g/l
Ht or HCT Hematocrit М 40-48% Newborns Reduced after 60
W 36-44% 44-64% years
children 35-45%
RDW Red Сells 11,5-14,5%
Distribution
Width
MCV Mean 83-98 mcm3 Newborns 128 fl Increasing after 50
Corpuscle 7 days 100-112 fl years (especially in
(Corpuscular) 6 months 78 fl smoking people) at
Volume 12 months 77-79 physiological age
fl iron-deficient ane-
4-5 months 80 fl mia - microcytosis
МСН Mean 27-33 pg 24-30 pg Less than norm
Corpuscular after 60 years (as
Hemoglobin anemia result)
MCHC Mean 32-36 g/dl More than norm Less than norm at
Corpuscular 320-360 g/l in newborns Fe-deficiency
Hemoglobin
Сoncentration
VSR or Velocity М 6-12 mm/h Newborns 1-2 1-2 mm/h
ESR sedimentation W 8-15 mm/h (at mm/h
rate or pregnancy up to 20 Up to 1 month 2-
erythrocytes mm/h as 6 mm/h
sedimentation hyperfibrino- 6-12 months 4-14
rate genemia result, mm/h
15-20 mm/h at 2-10 years 4-12
menstruation as mm/h
erythropenia
result)

127
ER Erythrocytic Min 0,42-0,48% Newborns: min In the old – both
resistance (the NaCl (hemolysis 0,48-0,52%, max min and max
most often beginning), max – – 0,24-0,30%; boarders get
osmotic one) 0,30-0,34% babies: min 0,46- decreased but
(complete 0,50%, max – minimal one –
hemolysis) 0,24-0,32%; 1-7 more significant
years min 0,46-
0,48%, max –
0,26-0,36%; 7-15
years min - 0,44-
0,48%, max –
0,28-0,36%
WBC White Blood 4,5-9,0 x 109/l Newborns 11,6- In the old – like in
Cells 20,6 the adult;
2 weeks 8,4-14,1 leucopeny – at
1 months 7,6 – purulent-septic
12,4 diseases, in
2 months 7,2- exhausted people,
11,6 at alimentary tract
6 months 6,7- diseases
11,3
1 year 6,8-11,0
7 years 5,9-9,3
15 years 5,5-8,5
WBC White Blood
Differen- Cells
tial Differential
GRA Granulocytes Other tables Often segmented-
(#) – absolute nucleated
numerals; % - (degenerative)
percentage shift to the right, it
correlation in is a sign of blood
leucocytic getting old
formule
Neut: Neutrophils 50-70%
1) Stabs or Rod-nucleated 1-4%
stab neutrophils
neutro-
phils, rods
or rod
neutrophils
2) Bands Segment- 50-65%
nucleated
neutrophils
Eos Eosinophils 1-4%
Bas (Baso) Basophils 0-1%

128
AGRA Agranulo- In deep old people
cytes there can be
monocytopeny and
lymphocyte-peny
Lymph Lymphocytes 20-40%:
T – 40-70%;
B – 20-30%;
0 (zero) or T- and
B- lymphocytes
predecessors – 20-
30%
Mon Monocytes 2-10%
(Mono)

Table 8
NEW-BORNS LEUCOCYTE FORMULE

Day Mye- Meta- Rods Segm Lymph Mon Eos Bas

locytes myelo-
cytes
1-st 0-4 0-4 0,5-11,3 51,4-72,0 16,1-33,3 3,1-9,5 1,0-5,0 0-1
hour
1 day 0-1,5 0-4 0,8-12,4 49,6-72,8 15,5-31,7 4,1-10,5 0,7-3,5 0-1
2 day 0-2,5 0-5 0,5-11,3 46,9-69,1 18,6-34,8 4,7-12,1 0,8-5,0 0-1
3 day 0-1 0-4 1,0-6,6 41,5-63,5 21,9-40,3 5,9-14,3 1,7-5,7 0-1
4 day 0-0,5 0-3 1,2-5,4 36,0-59,0 26,1-47,1 5,6-15,0 1,6-6,2 0-1
5 day 0-2 0-4 1,3-5,1 32,4-54,0 30,7-49,9 6,4-14,4 1,8-6,0 0-1
6 day 0-2 0-3 1,1-4,5 40,5-54,5 31,5-53,7 6,8-14,2 1,5-6,3 0-1
7 day 0-1 0-4 1,4-4,6 29,0-47,0 36,5-55,1 6,1-14,9 1,7-5,7 0-1
8 day 0-1 0-4 1,2-4,6 29,5-48,4 37,0-55,4 6,0-14,2 1,5-5,7 0-1
9-15 0-0,5 0-4 0,9-4,1 26,3-47,5 38,0-57,8 6,2-14,8 1,9-6,3 0-1
days
Table 9
BABIES LEUCOCYTIC FORMULE, %

Months Rods Segm Lymph Mon Eos

1 0,9-3,1 17-39 46-70 4,2-11,8 1,8-6,2
2 0,9-3,1 16-34 52-72 4,4-11,6 1-5
3 0,8-3,2 18-36 51-71 4-10 1-5
4 1,0-3,0 19-39 48-68 3,7-10,3 1-5
5 0.9-3,1 21-39 48-68 3,7-10,3 1-5
6 0,8-3,2 20-40 47-69 3,9-10,1 1-5
7 0,9-3,1 20-40 48-68 4-10 1,9-5,1
8 0,8-3,2 21-43 45-67 3,8-10,2 1-5
129
9 0,8-3,2 22-42 46-66 4-10 1-5
10 0,8-3,2 24-44 44-64 4-10 1,2-4,8
11 0,8-3,2 25-43 43-65 4-10 0,9-5,1
12 0,8-3,2 23-43 44-66 4-10 0,8-5,2

Table 10
DIFFERENTIATED LEUCOCYTES OF 2-15-YEARED CHILDREN, %

Years Segm Rods Lymph Mon Eos

2 1-3 28-48 37-61 5-9 1-7
3 1-3 32-54 34-56 4-8 1-7
4 2-4 34-54 33-53 4-8 2-6
5 1-3 35-55 33-53 3-9 2-6
6 1-3 38-58 30-50 3-9 2-6
7 1-3 39-57 32-50 4-8 1-5
8 1-3 41-59 29-49 4-8 1-5
9 1-3 43-59 30-46 4-8 1,5-4,5
10 1-3 43-59 30-46 4-8 1-5
11 1-3 45-57 30-46 3-9 1,5-4,5
12 1-3 44-60 29-45 4-8 1-5
13 1-3 45-59 30-44 4-8 1-5
14 1-3 46-60 28-44 4-8 1-5
15 1-3 45-61 29-45 3-9 1-5

PLATELETS FUNCTIONS ASSESSMENT CRITERIA

• platelets absolute quantity;
• capillary bleeding time;
• platelets aggregational activity;
• blood clot retraction;
• prostaglandines (thromboxanes, prostacyclines) metabolism.

COAGULOGRAM CHANGES IN CHILDREN

In mature new-borned
Vascular-platelet hemostasis:
• platelet amount is like in bigger children and even in the adult;
• bleeding duration is like in the adult;
• platelet clot retraction is like in the adult;
• platelets morphology is like in the adult;
• adhesive function is like in the adult;
• aggregation activity is reduced to ADP, collagen, epinephrine (due to weakened
releasing reaction, proaggregants decreasing in part thromboxan and
prostaglandins).
Coagulation hemostasis:
130
 • the XII-th factor level is equal to 20-70% from adult level (up to the 9-14th days
of life);
• prekallikrein level is reduced up to 20-50%;
• highly-molecular kininogen is lowered up to 40-80% of the adult level;
• thus, both blood coagulation and fibrinolysis are retarded;
• but even deep hypocoagulation is not accompanied by bleedings;
• this period distinguishing feature is vit K-dependent factors (II, VII, IX and X)
lowering especially on the 3rd and the 6th days of life (they come to norm up to the
14th day of life);
• early cutting of cord before so-called “placenta autotransfusion” id est blood
pumping from placenta vessels into the child blood stream leads to bigger
lowering in these vit K-dependent factors content;
• similarly, maternal breast early giving (during 2 first hours after birth) lowers
significantly mentioned factors depression after birth comparatively to breast later
giving (in 6-8 hours);
• antithrombin III level in plasma is lowered up to 50-69% and reaches its normal
level up to the 6th month;
• protein C and S contents are reduced significantly;
• plasminogen level is lowered up to 40-50% (up to the 6th month);
• plasminogen activator level is increased during the 1st 7 days;
• fibrinolysis activation is especially observed at cord cutting in 3-5 min (if right
after birth – in less extent);
• if coagulation factors level is less than 10% than the child can be determined to
the risk group on hemorrhagies;
• if coagulation factors content is more than 60% than the child can be determined
to the risk group on thrombosis.
But one should remember that all mentioned features are physiological norm sign
but not pathological one.

In immature new-borned:
• hemostasis bigger depression than in mature babies;
• lowering all procoagulants except fibrinogen and fibrinase;
• maximal hypocoagulative shift is observed during the 1 st day of life (in mature
babies – during the 3rd-5th days);
• early hypocoagulation reason – physiological jaundice, factors deficient synthesis
in liver;
• bigger deficiency of the XII-th;
• bigger deficiency of antithrombin III, protein C;
• bigger deficiency of fibrinolytic system components;
• all mentioned changes determine bigger rate both of hemorrhagic and thrombotic
changes, more often DIC-syndrome development;
• increase up to the adult norm appears in different terms.

131
 1-12-yeared children
• blood coagulation time reaches adult numbers up to an end of the 1st year of life;
• platelets number – is like in the adult;
• but many junior thrombocytes (8-17%, in the adult – 4-5%);
• 7-8% - old platelets;
• 10-11% - atypic forms;
• prothrombin content is fluctuated more both in the side of increase and decrease;
• the V-th and VII-th factors level is reduced comparatively to the adults;
• thus, there is big variety in indexes and they do not reach adult norms.

The biggest varieties –during puberty.

TOTAL BLOOD
Name_________________________________________
Sex________________
Age_______________
Special conditions (in part, pregnancy)________________
Er number________________________________
Hb level__________________________________
Color index_______________________________
VSR (ESR)_______________________________
Hematocrit_______________________________
MCV ____________________________________
MCH ____________________________________
MCHC__________________________________
Reticulocytes_____________________________
Leucocytes number( L)______________________
Junior (JUN) or bands ______________________
Stabs or rods_____________________________
Segments (SEGM) ________________________
Basophils (BAS) __________________________
Eosinophils (EOS) ________________________
Lymphocytes (LYMPH) _____________________
Monocytes (MON)__________________________
Platelets (PLT) ____________________________

4. Literature recommended:
1. Lecture course.
2. Mistchenko V.P., Tkachenko E.V. Methodical instructions for dental students
(short lecture course).-Poltava, 2005.-P.38-47.
3. Mistchenko V.P., Tkachenko E.V. Methodical instructions for medical students
(short lecture course).-Poltava, 2005.-P. 60-81.

132
 4. Mistchenko V.P., Tkachenko E.V. Blood system Physiology //Methodical
recommendations to practical classes for students of medical and dental
departments.-Poltava, 2005.-20p.
5. Stuart Ira Fox. Human Physiology.-8th Ed.-McGrawHill, 2004.-P.367-378, 444-
476.
6. Seeley R.R., Stephens T.D., Tate P. Essentials of Anatomy and Physiology.-The
3rd Ed.-McGraw Hill, 1999.-P. 286-304, 372-392.

LESSON 40
PRACTICAL SKILLS ON BLOOD SYSTEM PHYSIOLOGY

1. VSR (ESR) estimation methodics, the index norm.

2. Hemoglobin estimation methodics, the index norm.
3. Erythrocytes number estimation methodics, the index norm.
4. Leucocytes number estimation methodics, the index norm.
5. Coagulogram, main indexes norm.
6. Blood groups determining with Tsoliclons.
7. Blood groups determining with standard sera.

GLOSSARY
BLOOD SYSTEM PHYSIOLOGY
A
ABO system – the most common system of classification for red blood cell antigens;
on the basis of antigens on erythrocytes surface, individuals can be type A, type B, type
AB, or type O.
Acidosis – condition characterized by a lower than normal blood pH (pH of 7,35 or
lower).
Adaptive immunity – immune system response in which there is an ability to
recognize, remember and destroy a specific antigen.
Adhesion of platelets – the first stage of vascular-platelet hemostasis when platelets
are attached to injured place in vascular wall.
Agglutination – a clumping of cells (usually erythrocytes) as a result of specific
chemical interaction between surface antigens and antibodies.
Aggregation – making conglomerates.
Aggregation of platelets – the second stage of vascular-platelet hemostasis when
thrombocytes are united together and form conglomerate.
Agranulocytes – leucocytes with very small cytoplasmic granules that cannot be
easily seen with the light microscope; lymphocytes and monocytes.
Alkalosis - condition characterized by a higher than normal blood pH (pH of 7,45
or above).

133
 Allergy – a state of hypersensitivity caused by exposure to allergens; it results in the
liberation of histamine and other molecules with histamine-like effects.
Anemia – any condition that result sin less than normal hemoglobin in the blood or
a lower than normal number of erythrocytes.
Anisocytosis – different-sized erythrocytes (microcytes, normocytes, macrocytes)
simultaneous presence in one blood smear.
Antibodies – proteins found in the plasma that are responsible for antibody-
mediated (humoral) immunity; immunoglobulin proteins (G, M, D, E, A, D) secreted
by B-lymphocytes that have been transformed into plasmic cells (plasmocytes); their
synthesis is induced by specific antigens and they combine with these specific antigens
but not with unrelated antigens.
Antibody-mediated immunity – immunity resulting from B cells and the
production of antibodies.
Anticoagulant – chemical that prevents coagulation or blood clotting; an example
is antithrombin.
Antigen – a molecule able to induce the production of antibodies and to react in a
specific manner with antibodies.
Autoantibody – an antibody that is formed in response to, and that reacts with
molecules that are part of one's own body.
Autoimmune disease – disorder resulting from a specific immune system reaction
against self-antigens.
B
Basophil – the rarest type of leucocytes; a granular leucocyte with an affinity for
blue (purple) stain with basic dyes; promotes inflammation, participates in allergy and
prevents clot formation.
Basophylopeny – basophils number decreasing.
Basophyly – basophils number increasing.
Blood group – a category of erythrocytes based on the type of antigen on the surface
of the red blood cells; for example, the ABO blood group is involved with transfusion
reactions.
Buffer – chemical that prevents changes in pH when either an acid or base is added
to a solution containing the buffer; it realizes this by either combining with hydrogen
proton or by releasing hydrogen proton into solution.

C
Carbhemoglobin – hemoglobin compound with carbon dioxide.
Carboxyhemoglobin – hemoglobin compound with carbon monoxide the affinity
to which is higher in 450 times than to oxygen that defines strong intoxication.
Cell-mediated immunity – immunity resulting from the actions of T-cells.
Clot – to coagulate; a soft insoluble mass formed when blood coagulates.
Clotting factor – one of many proteins found in the blood in an inactive state;
activated in a series of chemical reactions that result in the formation of a blood clot.
Coagulation – the process of changing from a liquid to a solid, especially blood;
main stages in blood are following: prothrombinase forming (by extrinsic or intrinsic
way), prpthrombin transformation into thrombin (under prothrombinase action),
134
fibrinogen transformation into fibrin (under thrombin action), clot retraction (under
fibrinase action).
Complement – group of 9 serum proteins that stimulates phagocytosis,
inflammation and lysis of cells.
Constant region – part of an antibody that does not combine with an antigen and is
the same in different antibodies, responsible for activation of complement and binding
the antibody to cells such as macrophages, basophils, and mast cells.
Cyanosis – blue coloration of the skin and mucous membranes caused by
insufficient oxygenation of blood.

D
Deoxyhemoglobin – the form of hemoglobin in which the heme groups are in
normal reduced form but are not bound to a gas; deoxyhemoglobin is produced when
oxyhemoglobin releases oxygen.
Donor – a face giving part of his blood, bone marrow, other tissues or organ for
transfusion or transplanting to other person.
Duke probe – bleeding time from index finger or ear lobe at puncture making to the
depth equal to 3 mm; index of vascular-platelet hemostasis; norm is 2-4 min.

E
Embryonic stem cells – the cells of the inner cell mass of a blastocyst; are
pluripotent and so are potentially capable of differentiating into all tissue types except
the trophoblast cells of a placenta.
Endothelium – the simple squamous epithelium that lines blood vessels and the
heart.
Eosinophylopeny – eosinophils number lowering.
Eosinophils – granulocytic leucocytes with granules that stain red with acidic dyes;
are capable to phagocytosis, participate in allergy.
Eosinophily – eosinophils number rising; in part, it is observed at allergy and
helminthoses.
Erythroblast – immature erythrocyte, erythrocyte precursor.
Erythroblastosis fetalis – hemolytic anemia in a Rh-positive newborn caused by
maternal antibodies against the Rh factor that has crossed the placenta.
Erythrocyte – a red blood cell; erythrocytes are formed elements of blood that
represent biconcave discs, contain hemoglobin, transport oxygen and carbon dioxide;
do not have a nucleus.
Erythrocytosis – erythrocytes number increasing.
Erythrocytopeny – erythrocytes number decreasing.
Erythropoiesis – erythrocytes formation; it is realized by 2 ways – specific – due to
erythropoietin action; non-specific – due to hormones, vitamins and microelements
action.
Erythropoietin – the only specific regulator of erythropoiesis; protein hormone that
stimulates erythrocyte formation.
Extrinsic way of prothrombinase formation – Hageman-dependent, trigger
mechanism is hyperepinephrinemy; it is realized during blood coagulation.
135
 F
Fibrin – threadlike protein fiber derived from fibrinogen by the action of thrombin;
forms a clot, that is, a network of fibers that traps blood cells, platelets, and fluid, which
stops bleeding.
Fibrinogen – a protein in plasma that gives rise to fibrin when acted on by thrombin
to form a clot; serves a precursor of fibrin; also called as factor I; plasma without
fibrinogen is known as serum.
Fibrinolysis – the breakdown of a clot by plasmin.

G
Gamma globulin – a family of proteins found in plasma; immunoglobulin G.
Granulocytes or granular leucocytes – leucocytes named according to the
appearance, in stained preparations, of large cytoplasmic granules; neutrophils,
basophils, eosinophils.

H
Hematocrit – the ratio of packed red blood cells to total blood volume in a
centrifuged sample of blood, expressed as a percentage.
Hematopoiesis – production of blood cells.
Hemoglobin – the combination of heme pigment and protein within red blood cells
that acts to transport oxygen and (to a lesser degree) carbon dioxide; hemoglobin also
serves as a buffer.
Hemolysis – the rupture of erythrocytes.
Hemolytic disease of the newborn – destruction of erythrocytes in the fetus or
newborn caused by antibodies produced in Rh-negative mother acting on the Rh-
positive blood of the fetus or newborn.
Hemorrhage – rupture or leaking of blood from vessels.
Heparin – a mucopolysaccharide found in many tissues, but in greatest abundance
in the lungs and liver. It prevents blood coagulation (so-called primary anticoagulant).
Homeostasis – existence and maintenance of a relatively constant environment
within the body with respect to functions and composition of fluids and body tissues;
the dynamic constancy of the internal environment, the maintenance of which is the
principal functions of physiological regulatory mechanisms.
Humoral immunity – the form of acquired immunity in which antibody molecules
are secreted in response to antigenic stimulation (as opposed to cell-mediated
immunity).
Hypervolemy - rising in circulating blood volume.
Hypovolemy – lowering in circulating blood volume.

I
Immunity – the ability to resist damage from foreign substances such as
microorganisms and harmful chemicals such as toxins released by microorganisms.

136
 Immunoglobulins – proteins that have antibody functions and provide humoral
immunity; they belong to classes A, D, G, M, E.
Innate immunity – immune system response that is the same on each exposure to
an antigen; there is no ability to remember a previous exposure to a specific antigen.
Interferons – small proteins that inhibit the multiplication of viruses inside host
cells and that also have radioprotective, antitumorogenic and immunomodulative
properties.
Interleukins – biologically-active substances that participate in interleucocytic
integration (in part, during non-specific resistance and immune reactions).
Intrinsic way of prothrombinase formation – thromboplastin-dependent, trigger
moment is celluls membrane damage and thromboplastin formation; it is realized
during blood coagulation.

L
Lymphocyte – a type of mononuclear leucocyte; the cell responsible for humoral
and cell-mediated immunity.
Lymphocytopeny – lymphocytes number lowering.
Lymphocytosis – lymphocytes number rising.
Lymphokine – any of a group of chemicals released from T cells that contribute to
cell-mediated immunity.
M
Macrophage – any large mononuclear phagocytic cell that contributes to both
specific and non-specific immunity.
Megacaryocyte – a bone marrow cell that gives rise to blood platelets.
Methemoglobin – the abnormal form of hemoglobin in which the iron atoms in
heme are oxidized to the ferrous form (Fe has covalence not II but III) due to which
metHb is incapable of bonding with oxygen.
Microcirculative, primary or vascular-platelet hemostasis – is realized in shine
vessels or (together with blood coagulation) in large ones; there are 4 stages in it:
vascular spasm, platelets adhesion, platelets aggregation, plug retraction.
Microphage – basophil, eosinophil and neutrophil – little phagocytes present in
blood.
Monoclonal antibodies – identical antibodies derived from a clone of genetically
identical plasma cells.
Monocyte – a mononuclear, nongranular, phagocytic leucocyte that can be
transformed into macrophage.
Monocytopeny – monocytes number lowering.
Monocytosis – monocytes number rising.

O
Oncotic pressure – the colloid osmotic pressure of solutions produced by proteins;
in plasma it serves to counterbalance the outward filtration of fluid from capillaries
caused by hydrostatic pressure.
Osmolality – a measure of the total concentration of a solution; the number of moles
of solute per kilogram of solvent.
137
 Osmosis – the passage of solvent (water) from a more dilute to a more concentrated
solution through a membrane that is more permeable to water than to the solute.
Osmotic pressure – a measure of the tendency for a solution to gain water by
osmosis when separated by a membrane from pure water; directly related to the
osmolality of the solution, it is the pressure required to just prevent osmosis.
Oxyhemoglobin – a compound formed by the bonding of molecular oxygen with
hemoglobin.

P
pH scale – a measure of the hydrogen ion concentration of a solution; the scale
extends from 0 to 14,0; a pH of 7,0 being neutral. A pH of less than 7,0 – acidic, and a
pH of greater than 7,0 – basic.
Phagocytosis – process of ingestion and digestion by cells of substances such as
other cells, bacteria, cell debris, and foreign particles.
Pinocytosis – cell drinking; invagination of the cell membrane to form narrow
channels that pinch off into vacuoles; this permits cellular intake of extracellular fluid
and dissolved molecules.
Plasma – the fluid portion of the blood; unlike serum (which lacks fibrinogen),
plasma is capable of forming insoluble fibrin threads when in contact with test tubes.
Plasma cells – cells derived from B lymphocytes that produce and secrete large
amounts of antibodies; they are responsible for humoral immunity.
Plasmin or fibrinolysin – an enzyme that breaks down the fibrin in blood clots;
derived from plasminogen.
Plasminogen – fibrin precursor.
Platelet or thrombocyte – a disc-shaped structure, 2 to 4 micrometers in diameter,
derived from bone marrow cells called megacaryocytes; platelets circulate in the blood
and participate in vessels trophic function and hemostasis (both microcirculative and
blood coagulation).
Platelet plug – accumulation of platelets that stick to connective tissue and to one
another to prevent blood loss from damaged blood vessels.
Pluripotent - a term used to describe the ability of early embryonic cells to
specialize to produce all tissues except the trophoblast cells in the placenta.
Polycytemia – increasing in erythrocytes, leucocytes and platelets number;
sometimes in literature this term is used only for designation of only erythrocytes
number increasing (thus, as a synonyme of the term erythrocytosis).
Prostaglandins – class of physiologically-active substances first derived from
prosthate (it gave the name) present in many tissues; effects include vasodilation, pro-
and antiaggregation, stimulation and contraction of uterine smooth muscle during
menstruation and labor as well as promotion of inflammation and pain.

R
Reduced hemoglobin – hemoglobin with iron in the reduced ferrous state; it is able
to bond with oxygen but is not combined with oxygen; also called deoxyhemoglobin.
Reticulocyte – erythrocyte precursor; name depends on net presence as endoplasmic
reticulum.
138
 Reticulocytosis – reticulocytes number increasing.

S
Serum – the fluid squeezed out of a clot as it retracts; supernatant when a sample of
blood clots in a test tube and is centrifuged; serum is plasma from which fibrinogen
and other clotting proteins have been removed as a result of clotting.
Sickle-cell anemia – a hereditary autosomal recessive trait that occurs primarily in
people of African ancestry, in whom it evolved apparently as a protection (in the carrier
state) against malaria; in the homozygous state, hemoglobin S is made instead of
hemoglobin A, which leads to the characteristic sickling of red blood cells, hemolytic
anemia and organs damage.
Stem cells – cells that are relatively undifferentiated (unspecialized) and able to
divide and produce different specialized cells.
Suppressor T-cells – a subpopulation of T-lymphocytes that acts to inhibit the
production of antibodies against specific antigens by B-lymphocytes.

T
T-cell – a type of lymphocyte that provides cell-mediated immunity (in contrast to
B-lymphocytes that provide humoral immunity through the secretion of antibodies);
there are three subpopulations of T-cells: cytotoxic (killer), helper and suppressor.
Thrombin – a protein formed in blood plasma during clotting that enzymatically
converts the soluble protein fibrinogen into insoluble fibrin.
Thrombocyte – a blood platelet.
Thrombocytopeny – thrombocytes number lowering.
Thrombocytosis – thrombocytes number rising.
Thrombopoietin – a cytokine that stimulates the production of thrombocytes (blood
platelets) from megakaryocytes in the bone marrow.
Thrombosis – the development or presence of a thrombus.
Thrombus – A blood clot produced by the formation of fibrin threads around a
platelet plug.

W
White blood cells – see leucocytes.

TESTS ON BLOOD PHYSIOLOGY

1. Heparin sometimes is injected to experimental rats before blood taking if they

possess blood coagulation high degree. Why?
A. * Anticoagulation increasing.
B. Anticoagulation decreasing.
C. Coagulation increasing.
D. Fibrinolysis enforcement.
E. Fibrinolysis weakening.

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 2. Physiologists have established that RBC number in blood depends on bone
marrow functional state as well as RBC life duration. Mark average term of erythrocyte
life span:
A. * 120 days.
B. 70 days.
C. 50 days.
D. 150 days.
E. 220 days.

3. Blood was poured in a test-tube. Its coagulation time was assessed as 6 min. After
thrombus formation the test-tube was putted to the thermostate. It was estimated in 1
day that thrombus got destroyed. It was directly due to:
A. *Plasmins.
B. Kinines.
C. Kallikreins.
D. Heparin.
E. Antithrombins.

4. Circulating blood volume in 80 kg-weighted person after durable physical

overloading is 5,4 l, hematocrit – 50%, blood general protein – 80 g/l. These blood
indexes first of all are the result of:
A. *Water loosing while sweating.
B. Erythrocytes number increasing.
C. Proteins content increasing in plasma.
D. Circulating blood volume rising up.
E. Diuresis enforcement.

5. Total blood results in 12-yeared patient N. are as following as: eosinophils amount
is increased up to 12%. Mark the state this phenomenon can be observed:
A. *Ascaridosis.
B. General intoxication syndrome.
C. Pneumonia.
D. Immunodeficient state.
E. Acute respiratory viral infection.

6. Differentiated leucogram detection is one of important clinical investigated

methods. This index reflects:
A.* Leucocytes different sets percentage correlation.
B. Total leucocytes.
C. Granulo- and agranulocytes percentage correlation.
D. Lymphocytes percentage to total leucocytes amount.
E. Granulocytes percentage correlation.
7. Human being was poisoned with carbonic monoxide (CO). Mark changings in
blood at this:
A. *Carboxyhemoglobin formation.
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 B. Methemoglobin formation.
C. Carbhemoglobin formation.
D. Reducted hemoglobin formation.
E. Acidosis development.

8. Cyanides intoxication was detected while analysis of blood taking from corpse by
forensic doctor. Mark death reason:
A. *Methemoglobin formation.
B. Carboxyhemoglobin formation.
C. Carbhemoglobin formation.
D. Reducted hemoglobin formation.
E. Blood pH changings.

9. It is known that erythrocytes main function represents oxygen transport from

lungs to cells of all organism tissues. Mark erythrocytes compound providing this
process:
A.*Hemoglobin.
B. Albumins.
C. Globulins.
D. Enzymes.
E. ATP.

10. Anemia is developed after stomach pyloric part removal. Mark this disease
developmental reason in this case:
A. *Internal Kasl’s factor absence.
B. Vitamin D absorbtion disorder.
C. Vitamin C absorbtion disorder.
D. Iron absorbtion disorder in stomach.
E. Bone marrow dysfunction.

11. 150 mmol/l of NaCl (1 liter) was injected to the patient with blood loosing. Mark
index that will be changed first of all:
A.* Blood oncotic pressure.
B. Intercellular liquid oncotic pressure.
C. Blood osmotic pressure.
D. Intercellular liquid osmotic pressure.
E. Intracellular osmotic pressure.

12. The sportsman total blood results are the following: Er – 5,5 x 1012/l, Hb – 180
g/l, L – 7 x 109/l, neutrophils – 84%, basophils – 0,5%, eosinophils – 0,5%, monocytes
– 8%, lymphocytes – 27%. These indexes testify to stimulation first of all of:
A. *Erythropoiesis.
B. Leucopoiesis.
C. Lymphopoiesis.
D. Granulocytopoiesis.
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 E. Immunogenesis.

13. Blood group was detected in a woman during labor. RBC agglutination reaction
occurred with standard sera of the 0(I), A(II) and did not occur with standard serum of
the B(III). The investigated blood belongs to the group:
A. *B(III).
B. O(I).
C. A(II).
D. AB (IV).
E. None of them.

14. Blood typing was made in the pregnant. RBC agglutination occurred with
standard sera of the 0(I), B(III) and did not occur with standard serum of the A(II). The
investigated blood belongs to the group:
A. *A(II).
B. O(I).
C. B(III).
D. AB (IV).
E. None of them.

15. Blood typing has been made in 30-yeared man right before the operation. Blood
is Rh(+). RBC agglutination did not occur with standard sera of the 0(I), A(II) and
B(III). The investigated blood belongs to the group:
A. * O(I).
B. A(II).
C. B(III).
D. AB (IV).
E. None of them.

16. Vitamin K deficiency due to hepatic proteins synthesis after the disease in 16-
yeared guy will lead first of all to disorder in:
A.*Blood coagulation.
B. Erythrocytes sedimentation velocity.
C. Anticoagulants formation.
D. Erythropoietins synthesis.
E. Blood oncotic pressure.

17. Erythropoiesis is under control both of nervous and humoral factors –

erythropoietins. Which of inner organs participates mostly in this control?
A.*Kidneys.
B. Lungs.
C. Liver.
D. Alimentary tract.
E. Pancreas.

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 18. Doctor did not take into account at citrate blood hemotransfusion that such blood
does not coagulate and bleeding is not interrupted. What was not taken into account by
the doctor?
A. *Ca++-ions absence in such a blood.
B. K+-ions absence in such a blood.
C. Na+-ions absence in such a blood.
D. Mg++-ions absence in such a blood.
E. Any answer is correct.
19. Er content is a healthy person blood is 5,65 x 1012/l. The reason of this can be
the fact that the investigated person:
A.*Lives in highland.
B. Is a miner.
C. Is the pregnant.
D. Is the adult.
E. Is a child of pre-school age.

20. Fibrinogen level in plasma in the pregnant was twice bigger than norm. Mark
VSR probable values:
A. *40-50 mm/h.
B. 10-15 mm/h.
C. 2-12 mm/h.
D. 5-10 mm/h.
E. 0-5 mm/h.

21. Heart activity stoppage appeared in a patient as a result of citric blood great
amount transfusion. Indicate to probable mechanism of received changings:
A.*Ca-ions lack in blood.
B. Iron excess.
C. Fibrinogen lack in blood.
D. Er excess.
E. Circulating immune complexes.

22. The patient complaints on durable bleeding at insignificant traumatic lesion.

Blood laboratory analysis showed blood content disorder. Which of cells get in touch
with this?
A. *Platelets.
B. Erythrocytes.
C. Neutrophils.
D. Lymphocytes.
E. Monocytes.

23. Doctor has performed workers’ examination for assessment their adaptation to
physical loading after hard overloadings performance. What changings in total blood
can be revealed?
A.*Redistributive leucocytosis.
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 B. Leucopeny.
C. Anemia.
D. Hypoalbuminemia.
E. Differentiated leucogram shift to the left.

24. The patient H. is having been sick for bronchial asthma for 15 years. What
changings in differentiated leucogram can be found by the doctor in his leucogram?
A.*Eosinophyly.
B. Basophyly.
C. Leucocytosis.
D. Leucopeny.
E. Leucocytic formule shift to the left.

25. Blood in vascular bed is in liquid state under usual conditions. It is provided by:
A.*Vessels endothelium smooth surface.
B. Fibrin thin layer presence on vascular wall.
C. Prostacyclin synthesis with endotheliocytes.
D. Similar charge of vascular wall and blood formed elements.
E. All the mentioned factors.

26. 2 guys (18-yeared, 180 m in height) were examining. Body mass of one guy was
56 kg, the second one – 96 kg. Stress reaction was caused in them for autonomic
nervous system disorders diagnostics. How blood coagulation will be changed in them?
A.*Will be without changings in the first one and will grow in the second one.
B. Will grow in the second one and will be without changings in the first one.
C. Will be decreased in the first one and will be without changings in the
second one.
D. Will be without changings in the first one and will be reduced – in the
second one.
E. Will be without changings in both.

27. Which of mentioned statements is true?

A.*Hb is in erythrocytes in healthy adult person blood.
B. WBC content is bigger than RBC in healthy adult person blood.
C. Platelets content is bigger than RBC in healthy adult person blood.
D. Lymphocytes level is more than the one of neutrophils in healthy adult
person blood.
E. Eosinophils are the most widely-spread leucocytes set.

28. There is heparin in basophils granules. Which of anticoagulants activity is rised

due to heparin action?
A.*Antithrombin III.
B. Antithrombin I.
C. Fibrin.
D. Prothrombin.
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 E. Fibrinogen.

29. Hepatocytes toxic injury with protein-synthesizing function in a patient is

accompanied by albumins content as well as oncotic pressure significant decreasing in
blood plasma. What phenomenon will be these changings results:
A. *Edemas appearance.
B. Diuresis decreasing.
C. ESR (VSR) decreasing.
D. Blood volume increasing.
E. Blood density increasing.

30. Gums disease is in a man. He has gums hyperemy due to microcirculative bed
afferent vessels dilation. What leucocytes substance provided mentioned changings:
A. *Histamine.
B. Substance P.
C. Epinephrine.
D. Endorphins.
E. Acetylcholine.

31. Girl complaining on spontaneous (without traumas and other injuries) frequent
subcutaneous hemorrhagies which do not disappear then for long. Blood analysis
showed that platelets number decreasing up to 60x109/l. Platelet decreased number will
be reflected to:
A. *Vessels endothelium trophycs.
B. Gases transport with blood.
C. Blood phagocytic peculiarities.
D. Blood osmotic pressure support.
E. Acid-alkaline equilibrium.

32. 27-yeared man addressed the doctor with complains on coagulation time
increasing up to 15-18 minutes. What coagulation factor deficiency increases
significantly platelets irreversible aggregation phase:
A. *Тhrombin.
B. Fibrinogen.
C. Аntihemophilic globuline В.
D. Аntihemophilic globuline С.
E. Кininogen.

33. Blood coagulation represents consequent enzymatic process. Indicate to

vascular-platelet hemostasis phase Willebrand factor influences on:
A .*Platelet adhesion.
B. Vessels reflectory spasm.
C. Platelets reversible aggregation.
D. Platelets irreversible aggregation.
E. Platelet plug retraction.
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34. Which of mentioned functions is not major for blood plasma:
A.. *О2 transporting.
B. Hormones transporting.
C. Red blood cells sizes support.
D. Chilomicrones transporting.
E. Antibodies transporting.

35. Hematocrit 41% means that in one blood unit:

A. *Erythrocytes, leucocytes and platelets represent 41 % from total volume.
B. 41 % of hemoglobin is present in plasma.
C. Blood plasma represents 41 % from total volume.
D. 41 % of hemoglobin is present in erythrocytes.
E. 41 % of blood elements are erythrocytes.

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