MINISTRY OF HEALTH OF UKRAINEMINISTRY OF EDUCATION AND SCIENCE OF

UKRAINESUMY STATE UNIVERSITY

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

PHYSIOLOGY OF THE BLOOD

Educational book

Is recommended by the Central methodological studywith higher education the Ministry of Health of Ukraine

SumySumy State University

2010

3

UDK 612.1BBK 67.9 (4УКР) я7 G72

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

Is recommended by the Central methodological study with higher educationthe Ministry of Health of Ukraine as educational bookfor students of higher education IV level accreditation

(letter № 23-01-25/114 from 28.10.2009)

Garbuzova V.Yu., G72 Physiology of the blood: educational book / V.Yu. Garbuzova, L.А. Los, O.A. Obukhova – Sumy: Publisher SumSU. – 2010. – 165 p. ISBN 978-966-657-272-4

This teaching aid described in the tutorial material for self-prepar-ing students to practical classes in physiology from the “Physiology of the blood”. Each theme contains basic information theoretic question to self, test questions and problems.

For students of institutions of the III - IV level of accreditation.

У навчальному посібнику викладений матеріал для самостійної підготовки студентів до практичних занять з фізіології з розділу “Фізіологія крові”. До кожної теми наведені основні теоретичні відомості, питання до самопідготовки, тестові питання і задачі.

Для студентів вищих медичних закладів III – IV рівнів акредитації.

4

CONTENTS

INTRODUCTION…………………………………………… 4

Objectives……………………………………………………. 5

Chapter 1 Physical and Chemical Properties of the Blood….. 6

Chapter 2 Physiologies of Erythrocytes……………………… 34

Chapter 3 Blood groups…………………………………….... 62

Chapter 4 Protective functions of the blood. Leucocytes……. 85

Chapter 5 Haemostasis……………………………………...... 111

PRACTICAL WORKS……………………………………..... 138

Appendix I…………………………………………………… 163

Appendix II………………………………………………….. 165

5

INTRODUCTION

Blood is used to transport substances and together with lymph and intercellular fluid belongs to the internal medium of the organism. With the help of well regulated constant content and properties of it, the internal medium provides comparably independent existent of the organism in conditions of the envi-ronment. Compounds of internal medium have common and differential physical and chemical properties. They can affect each other, and their state depends on activities of various sys-tems of the organism.

System of the blood is the variety of executive organs (blood, which is circulated and stored; organs of blood forma-tion and blood degradation) and mechanisms of regulation (nervous and humoral), activity of which is directed on keeping the adequate changes of blood compounds to provide adaptive reactions.

Blood participates in transport of substances, helps the excretion of metabolic products, it provides protection from antigens and non-protein factors, affects the regulation of dif-ferent functions of the organism.

Chapter “Physiology of the blood” is important in prepar-ing the medical doctor of any specialty, because the state of in-ternal medium describes all processes in organism, which char-acterize homeostasis, homeokinesis and adaptive reactions when changes in internal medium and co-operation of the or-ganism with the environment.

Teach chapter “Physiology of the blood” is necessary for learning the next chapters of physiology and other subjects, for example pathological physiology and all clinical medically-specified subjects.

6

Objectives:

To know the definition of blood system, mechanisms of its regulation based on analysis of homeostasis parameters: blood volumes, acid-base balance, osmotic pressure, quan-tity and quality content of blood plasma and formed ele-ments of the blood.

To know physiological laws of functions of blood system: respiratory, transport and protection.

To know physiological laws of functions of keeping the liquid consistency of the blood and development of hemostasis when damage of blood vessels.

To make conclusions about the state of physiological func-tions of the organism, which perform with the help of the blood system, based on quality and quantity showings: hematocrit, number of erythrocytes, hemoglobin, leuco-cytes, thrombocytes, leukogram, color index, erythrocyte sedimentation rate (ESR), time of erythrocytes coagula-tion, duration of bleeding.

To analyze maturing changes of blood consistence, func-tion and mechanisms of regulation.

To explain the physiological basis of examination methods of functions of the blood system: quantity of formed blood cells, hemoglobin, ESR, osmotic resistance of erythro-cytes, duration of bleeding, time of blood clotting, defini-tion of blood group in ABO and CDE.

7

CHAPTER 1 Physical and Chemical Properties of the Blood

Blood is one of many functional systems of the organism. Together with nervous system blood unties organs in entire or-ganism. At the same time they define the term – proper blood system or physiological blood system (PBS).

The contents of PBS are:1 Peripheral blood.2 Organs or blood formation and blood degradation.3 Mechanisms of nervous and humoral regulation of blood

content.

Functions of the Blood

Functions of the blood are important and various. Practi-cally all of them are associated with the circulation of the blood in blood vessels. That is why the main function of the blood is transport function. There are several types of it:1 Respiratory function (transport of gases: O2 from the lungs

to tissues, CO2 from tissues to the lungs).2 Trophic function (transport of nutrients from gastro-intesti-

nal tract and other organs to all the tissues of the organism).3 Excretory function (transport of metabolic products to the

excretory organs).4 Regulatory function (transport of hormones and biologically

active substances from endocrine glands to aim-organs).5 Protective function (transport of phagocytes and im-

munoglobulins).6 Thermoregulatory function (transport of heat from organs

that maintain warmness – liver and other internal organs to the skin).

8

Besides, pathogenic factors are transported with blood: microorganisms, toxins, tumor cells. Transport of the last one will lead to the development of metastasis of malignant tumor.

Except the transport function, blood plays an important role in maintaining homeostatic features of the organism. That is why the second important function of the blood is homeo-static function. There are several types of it:1 Maintenance of the constant chemical content and physical

properties of the blood (osmotic pressure, pH, temperature, concentration of ions and other).

2 Maintenance of the constant volume of the circulating blood.

3 Maintenance of the antigenic homeostasis.The third, important function of the blood is creative

function. Macromolecules, which are transported with blood, perform intercellular information transferring, which provides regulation of intracellular processes of protein synthesis, keep-ing the level of cell differentiation, renovation and maintenance of tissue structure.

Content and Quantity of the Blood

Peripheral blood – is the blood, which circulates in ves-sels and it is stored.

The volume of circulating blood (VCB) is 6-8% of body weight of adult human or 70 – 75 ml/kg of the body weight (approximately 4 – 6 liters).

VCB is an important physiological constant. VCB de-pends on:1 Age (VCB of the newborn is 10% of the body weight and

only in the period of pubescence it decreases to the level of the adult).

2 Sex (men – 7-8%, women – 6-7% of the body weight).

9

3 Functional state of the organism (physically trained have higher VCB, sportsmen can have up to 10%).

Normal VCB value is normovolemia, increased VCB – hypervolemia, decreased VCB – hypovolemia.

Peripheral blood consists of plasma (55 – 60%) and formed elements (40 – 45%) fig. 1.1.

Percentage volume of the formed elements is called hematocrit. In normal state hematocrit value is HV almost completely depends on the quantity of erythrocytes in the blood, because its volume is nearly 99% of the volume of all formed elements of the blood. Only in some forms of leucosis, due to development of anemia and increase in quantity of cir-culating leucocytes, the part of the last ones in hematocrit value increases.

HV = (Red Cell Volume Total blood Volume) 100

Hematocrit is defined by Wintrobe method. 2 ml of the blood is put in special centrifugal glass, then add anticoagulant to it and turn it for 10 minutes with 1000 turns per minute. Blood cells, mass of which is higher than plasma will settle down in the bottom. Due to plasma are lighter in weight than erythrocytes, they will form thin white layer between erythro-cytes and plasma.

Plasma

Leucocytes

Erythrocytes Thrombocytes

Figure. 1.1 – Compaund of the blood

10

Other method for determination of haemotocrite value is microtechique: Blood sample is withdrawn in a heparinized capillary glass tube of small size. Both ends are waxed. The tube is put with other similar tubes in a special centrifuge and rotated for few minutes. The tube is then removed after separa-tion of red cells from plasma and put on a special scale to read the haematocrite value.

Hematocrit value depends on:1) sex (men – 44% - 46%, women – 41% - 43%);2) age (newborns have 20% higher, than women; children –

10% higher);3) life conditions (due to adaptation to the mountain country,

hematocrit can increase);4) HV is greater in venous than arterial blood (due to chloride

shift phenomenon) and in large, than in small vessels.The increase of hematocrit can lead to increase of blood

stickness and that means to increase of the load on heart, disor-ders of blood circulation.

Changes in haemotocrite value (HV)1) HV is increased in:

a) polycythaemia: due to increased number of R. B. Cs as in high altitude and in the newly born infants;

b) dehydration: due to decreased plasma volume as in severe vomiting and diarrhea.2) HV is decreased in:

a) anemias: due to decreased R. B. Cs. count;b) Overhydration: as in renal diseases or after intravenous

infusion of large amounts of fluids (fig. 1.2).Uses of haemotocrite value:

1) diagnosis of anaemias;2) determination of blood volume and renal blood flow;3) calculation of certain blood indices;4) follow up of cases of shock.

11

Functional importance of blood plasma components

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

Main components of blood plasma are: Water (91%); Proteins (8%); Electrolytes (0,9%).

Importance of Water

1 Water medium, in which substances and blood cells are dis-solved.

2 Water defines VCB.

12

3 It is needed to perform the exchange of the substances be-tween blood and intercellular fluid.

4 It affects reological properties of the blood (for example, stickness).

5 Due to high thermal capacity it performs transport of heat.

Importance of Proteins

1 Transport function. There are special spots in the molecule of protein, which are able to bind inorganic substances (for ex-ample, ions, water) and organic substances (for example, hor-mones, biologically active substances) and to transport them. Binding of this substances to proteins provides:

a) Keeping small molecules in bloodstream when blood is passing through kidneys.

b) Predicts its destructing by blood enzymes.There are specific and non-specific transport proteins.

Not-specific – are able to bind different substances and trans-port those (most of albumins transport hormones, calcium). Specific – transport only one kind of substances. For example, cerulloplasmin – ions of cuprum (Cu), transferrin – ions of iron (Fe), haptoglobulin – bilirubin.2 Trophic function. Proteins are the source of amino acids,which with peripheral tissues, are used for the formation of proper, specified for the organ proteins. Proteins are the source of energy. When breaking down 1 g of protein in the organism 4,1 kkal.

Trophic function of proteins is used clinically when dis-order of natural way of nourishment, in parenteral nourishment, when protein suspensions are injected in bloodstream.3 Enzymatic function. There are a lot of protein-enzymes into the plasma. There are secretory and indicatory (cellular) en-zymes. Secretory enzymes are synthesized in liver and are ex-creted in the blood plasma, where they perform their function.

13

Typical representatives of this group are protein-enzymes of blood clotting. Indicatory enzymes are entering the blood from other organs. Their activity is not high. In conditions of patho-logical states, enzymes are taken from cells into the blood and its activity increases, which indicate the level of lesion. That’s why quantitative definition of blood enzymes is one of the available laboratory methods of diagnostics. For example, ac-tivity of AlAt (alaninaminotransferase) increases during liver sickness. Activity of AsAt (aspartateaminotransferase) in-creases up to 20 times during myocardial infarction. Activity of lactatedehydrogenase increases during myocardial infarction, hepatitis, myopathy, tumors, and leucosis.4 Participating hemostasis. Proteins comprise biochemical systems of blood plasma, which provides hemostasis, namely:

Blood clotting system; Anticoagulatory system; Fibrinolytic system; Kallikrein-kinine system.

5 Participating in maintaining pH of the blood. Proteins form protein buffer. In acidic medium, they work as bases, binding acids; in base, they react like acids, binding bases. This prop-erty of the proteins is called amphoteric. Mostly buffer proper-ties belong to carboxyl groups and amino groups. Plasma pro-teins are responsible for 15% of the buffering capacity of the blood and carriage of CO2.- Protein – NH2 +CO2 NHCOOC (Carbamino protein);- Proteinic acid: Na proteinate buffer:

a) Na proteinate +H2CO 3 NaHCO3 + Proteinic acid.b) Na proteinate + lactic acid Na lactate + Proteinic acid.

Lactic acid (strong acid) is converted to proteinic acid (weak acid).6 Maintenance of the reological properties of the blood, namely its stickness. When increase in protein quantity, stick-

14

ness increases, when decrease in protein quantity, stickness de-creases.7 Proteins are the source of biologically active substances.For example, kinins and angiotensin.8 Protective function. Proteins participate in non-specific and specific protection of the organism. Non-specific protection is represented by complement system proteins, interferons, oroso-mucoid and viruses’ inhibitors. Specific – by antibodies: con-genital (agglutinins) and acquired.9 Making creative connections. Proteins participate transfer-ring of the information, which affects genetic apparatus of cells, provides growth, development and differentiation of tis-sues. For example, proteins are growth factor of nervous tissue, erythropoietin etc.10 Capillary Permeability. Plasma proteins close the pores in the cement substance (between the endothelial cells) of the capillary wall. Hypoproteinaemia increases the capillary per-meability. 11 Creation of oncotic. (colloid-osmotic) pressurePonc= 25 – 30 mmHg. 80% of oncotic pressure is made by albu-mins (molecule of albumin has small size and in volume of plasma, its quantity is the highest).

Role of oncotic pressure in redistribution of water in the organism (fig. 1.3):

15

C a p i l l a r y

Arterial part Venous part

Рhbp = 32,5 mmHg Рhbp = 17,5 mmHg

Рobp = 25 mmHg Рobp = 25 mmHg

Filtration Reabsorbtion

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

Рhp = 3 mmHg Рhp = 3 mmHgРop = 4,5 mmHg Рop = 4,5 mmHg

Figure 1.3 – Redistribution of water in the capillaries

Wall of capillaries is permeable to small molecules and water. That is why osmotic pressure in blood plasma and in intercellular liq-uid is almost equal. Big molecules, first of all protein molecules, can not pass through the capillary wall. That’s why there is gradient of protein concentration (oncotic pressure gradient - Ponc) between plasma and intercellular liquid. Ponc inside capillary is higher than in intercellular liquid. Hydrostatic pressure – pressure of the liquid on the capillary wall (from one side blood is making pressure on capil-lary wall, from other side – intercellular liquid), it also plays impor-tant role in redistribution of the water. Hydrostatic pressure of the blood is higher, than hydrostatic pressure of intercellular liquid.

16

Water exchange occurs in two ways:1) filtration (transition of the water from the capillary to the tis-

sue);2) reabsorbtion (transition of the water from the tissue to the

capillary).The direction of the water movement is defined by the

filtrative pressure (Pf).Pf = (Phbp + Pop) – (Php + Pobp)Phbp – hydrostatic pressure of the blood;Pop – oncotic pressure of the intercellular liquid;Php – hydrostatic pressure of the intercellular liquid;Pobp – oncotic pressure of the blood.If Pf >0 – filtration occurs.If Pf <0 – reabsorbtion occurs.

Increasing of the Phbp and Pop leads to filtration, increasing

of the Php and Pobp leads to reabsorbtion.In the arterial end of the capillary:

Pf = (32,5 + 4,5) – (25 + 3) = 9 mmHg – filtration occurs, water is transited to the tissue.

As the blood passes in the capillary, in the result of tran-sition of the water to the tissue, hydrostatic pressure decreases. In the middle of the capillary Pf = 0 and water transition stops.

In the venous end of the capillary:Pf = (17,5 + 4,5) – (25 + 3) = - 6 mmHg – reabsorbtion occurs, water passes into the capillary.

At the beginning of the capillary approximately 0,5% of blood plasma passes to the tissues. Pf in the arterial part of the capillary (Pf = 9 mmHg) is higher than in the venous part (P f = - 6 mmHg), that is why the bloodstream returns not the whole 100% of the liquid, but nearly 90%. 10% are excreted through the lymphatic vessels.

This pressure values may vary in different organs and it depends on organ activity. Described mechanism of filtration – reabsorbtion is called Starling’s mechanism.

17

Changes in any of the parameters may cause disorder in filtration and reabsorbtion correlation.

For example, the decrease in protein concentration in blood plasma will lead to the decrease of the reabsorbtion, de-lay of the water in intercellular medium and development of the intercellular oedema. This can happen during starvation (cachexy oedemas); when pathological processes in kidneys, in the consequence of which proteinuria can occur and loss of proteins (nephrotic oedemas); when disorder in albumin syn-thesis by liver (hepatic oedemas); when allergic and inflamma-tory processes, when there is the increase of vessel wall perme-ability and other plasma proteins are leaving to the intercellular space (membranogenic oedemas) and other.

Plasma protein consistency

Plasma proteins can be divided into several fractions:1 Albumins (35-50 g/l). They are mostly small proteins; their molecular mass is not more than 70000 Da, Synth esized by the liver only. They are circulating in the bloodstream for a long time: the period of halfexcretion is 10-15 days. The main func-tions of albumins are transport and trophic. Due to high hy-drophilic, small sizes and high concentration in blood plasma albumins play important role in the creation of oncotic pres-sure. Decrease in albumin concentration to 30 g/l and lower can lead to the decrease of oncotic pressure and the develop-ment of oedemas.2 Globulins (20-40 g/l): α1, α2, β, γ. Molecular mass is 44000-130000 Da, synthesized by the liver and reticuloendothelial cells as well as plasma cells present in the lymph nodes, spleen, bone marrow and liver. The term of circulation of globulins is less than the one of albumins: the period of halfexcretion is less than 5 days. The main functions of globulins are transport and protective.

18

3 Fibrinogen (2-4 g/l). It is the largest protein of blood plasma. Synth esized by the liver only. Fibrinogen plays a role in processes of blood clotting and thromb formation.

The correlation between albumins and globulins is called albumin-globulin coefficient or albumin – globulin ratio. Nor-mally it is 1,5 - 2,3.

Clinical importance of Albumin – Globulin ratio (A/g ra-tio):1 A/g ratio is decreased in:

a) liver diseases (e.g.hepatitis) due to decreased albumin for-mation;

b) renal diseases (e.g. nephrosis) due to albumin loss in urine;

c) infections and allergy due to increased synthesis of - globulins.2 A/g is increased in:

a) hypogammaglobulinaemia;b) acquired immunodeficiency syndrome (AIDS) due to de-

crease - globulins.

Importance of Electrolytes

The main function of blood plasma electrolytes is the cre-ation of osmotic pressure. Posm = 7,5 atm (0,3 osmole, 745 kPa, 5600 mmHg). The osmotic pressure is defined by the quantity of solved particles, not by the size of them. 96% of the osmotic pressure is made by Na+ and Cl- ions, because the molecular mass of NaCl is small.

Solutions, the osmotic pressure of which is the same as blood plasma has, are called isotonic. For example, 0,9% solu-tion of NaCl, Ringer’s solution, Ringer-Lock’s solution, Tirode, 5% glucose solution, haemodesis. Solutions with the osmotic pressure of which is higher than the plasma is called hypertonic, if it is lower it is called hypotonic.

19

In hypertonic solution water is leaving the cells, cells firm, their normal turgor is disbalanced. It is called plasmoly-sis. In clinics it is used for counting the erythrocytes: blood is diluted by 4% solution of NaCl, erythrocytes are firmed and they are easy to count.

In hypotonic solution water enters cells, cells swell, oedema of the cells occurs and destruction of the cells, which is called haemolysis. In both cases cells metabolism disorder oc-curs or even can lead to the death of the cell.

Plasmolysis Haemolysis

Figure 1.4 – Plasmolysis and haemolysis in erithrosyts

Demands to blood substitutes1 Isotonicity. Osmotic pressure of blood substitutes should be

equal to Posm of plasma. 0,9% solution of NaCl is the sim-plest blood substitute.

2 Balanced content of inorganic salts.3 Isooncoticity. Big molecules are slowly excreted from the

bloodstream, it helps extended refresh of volume of circulat-ing blood (VCB). This is reopolyglukin, haemodesis, poly-desis.

4 pH should be equal to the pH of blood plasma.5 Sterility.6 It should be non toxic.

Classification of blood substitutesGroup 1 – hemodynamical:

Low molecular dextranes – reopolyglukin;

20

Medium molecular dextranes – polyglukin;Gelatin substances – gelatinol.

Group 2 – disintoxicational:Low molecular polyvinylpyrolidon – heamodesis;Low molecular polyvinyl alcohol – polydesis.

Group 3 – preparations for parenteral nourishment:Protein hydrolysates – casein hydrolysate,Aminopeptide, aminokrovin, aminosol, hydrolysin;Solutions of amino acids – polyamine, maryamin, freeamin etc;Fatty emulsions – intralipid, lipofundin;Sugars and polybasic alcohols – glucose, sorbitol, fruc-tose.

Group 4 – regulators of fluid-electrolyte and acid-base balance: saline solutions – isotonic solution of sodium chloride, Ringer’s solution, lactosol, solution of sodium hydrocar-bonate, trisamin solution, etc.Functional system, which provides constancy of the os-

motic pressure.Osmotic pressure is an important physiological constant.

Any deviation of osmotic pressure from normal will lead to re-distribution of water between cell and intercellular medium.

For maintenance of the osmotic pressure on the same level in the organism there is a functional system, which con-sists of external and internal part. In basis of external part there are behavioral responses, which are responsible for normaliz-ing of osmotic pressure. If there is increase in osmotic pressure, human feels thirsty and drinks water. If there is decrease in os-motic pressure, human will feel to eat something salty. In basis of internal part there are local mechanisms of reflexes. Local mechanisms are the processes, which occur in blood itself. If decrease of osmotic pressure, excess water is connecting to the low molecular proteins, formed blood cells and Posm increases. If increase of osmotic pressure, excess electrolytes salts is ab-

21

sorbed by formed blood cells and transported to organs (K+, Ca2+ - to muscles, Ca2+, PO4

3- - to bones, Na+, Fe2+ - to the liver, Na+, Cu2+ - to the spleen, Na+, Ca2+, Zn2+ - to the pancreas) and Posm decreases.

These mechanisms are working during few hours and if Posm will not go to normal, local mechanisms of reflexes will start working.

There are 3 reflexes for the maintenance of Posm:1) osmoregulative;2) volumeregulative;3) Na-uretive.

Osmoregulative Reflex

When increase of osmotic pressure osmoreceptors are ir-ritated (peripheral – in vessels, heart, liver and central – in hy-pothalamus).

From osmoreceptors impulses are passing to supraoptical and paraventricular nuclei of hypothalamus, where ADH (an-tidiuretic hormone is formed). It is transported to the neurohy-pophysis through axons and it is released into the blood. It is the main hormone, which retain water in the organism.

Its targets are distal tubules and collecting tubules of the nephron. In distal tubules ADH interacts with α-receptors on basolateral cell membrane, excretion of hyaluronidase from cells activates, which breaks down hyaluronic acid from inter-cellular space, in result the permeability of epithelium of tubules for water increases. In tubules, ADH interacts with V2-receptors, adenylcyclase system activates, formation of cAMP increases, then cAMP diffuse in apical membrane, which acti-vates membrane permeability to water, ADH interacts with V1

in blood vessels. It leads to formation of inositol-tri-phosphate (ITP) and diacylglycerol (DAG), decrease of cAMP amount

22

and constricting of vessels. Due to this effect, there is another name of hormone – vasopressin.

Decrease of osmotic pressure is caused by decrease of the sodium level in blood plasma. Hyponatriemia stimulates secre-tion of renin. Renin activates formation of angiotensin II, which stimulates secretion of aldosterone by adrenal glands cortex. The targets of aldosterone are distal tubules of nephron, where it increases reabsorbtion of sodium ions, restores normal sodium concentration in plasma. In result, osmotic pressure in-creases.

Volumeregulating Reflex

This reflex starts working when VCB decreases by 7 – 15% (this change goes with increase of Posm). Excitation of vol-umereceptors in vessels, internal organs and cavities occur. From volumereceptors, impulses are passing to hypothalamus to supraoptical and paraventricular nuclei. Here ADH is formed, which increases reabsorbtion of water in kidneys and causes increase of VCB and decrease of Posm.

Na-uretive ReflexThis reflex works when VCB increases. In result volume

of blood that is passing to the heart, increases. Atrium walls overexpand. In the consequence of this myoendocrine cells of both atria release Na-triuretic hormone (atriopeptin). Target of this hormone is distal tubules of kidneys, where it decreases re-absorbtion of sodium, and, as a consequence, Na-uresis, diure-sis increase, VCB decreases, Posm increases.

Regulation of Secretion

Four important factors are involved in the regulation of secretion of aldosterone. The stimulatory agents for aldosterone secretion are:

23

1) increase in potassium ion concentration in the extracellular fluid; 2) decrease in sodium ion concentration in the extracellular fluid;3) decrease in extracellular fluid volume;4) adrenocorticotropic hormone.

Mechanism

Increase in the concentration of potassium ions is the most effective stimulant for aldosterone secretion. It acts di-rectly on the zona gromerulosa and increases the secretion of aldosterone. Decrease in sodium ion concentration and extra-cellular fluid volume stimulates the juxtaglomerular apparatus in the kidney to secrete renin. Renin acts on angiotensinogen in the plasma and converts it into angiotensin I, which is con-verted into angiotensin II. Angiotensin II acts on the zona gromerulosa to secrete more aldosterone. Aldosterone in turn, increases the retention of sodium and water. This causes an in-crease in the sodium ion concentration and volume of extracel-lular fluid.

Now, the increased sodium ion concentration and the ex-tracellular fluid volume inhibit the juxtaglomerular apparatus and stop the release of renin. So, angiotensin II is not formed and release of aldosterone from adrenal cortex is stopped.

Adrenocorticotropic hormone mostly stimulates the se-cretion of glucocorticoids more than aldosterone. Still, it has mild effect in stimulating aldosterone secretion.

Physical and Chemical Properties of the Blood

1 Osmotic pressure. Posm = 7,5 atm2 Density. Defined by presence of soluble substances.

pof plasma = 1,025 – 1,034 g/cm3

24

pof blood = 1,050 – 1,060 g/cm3

3 Stickness. Stickness – internal friction, which is made by friction of formed elements between each other and with vessel wall. Stickness resistance to bloodstream. Stickness of the liquid is defined comparatively to water, stickness of which is 1.

Stickness of plasma = 1,7 - 2,2Stickness of blood = 5.

Factors, which affect stickness:

1) hematocrit (the higher number of erythrocytes - the higher the stickness);

2) the amount of proteins (the higher amount of proteins - the higher the stickiness).

4 Active reaction of the blood (pH).pH of the blood – is reversed logarithm of the concentra-

tion of H+ ions. pH is determined by correlation of H+ ions and OH- in blood.

pH of the arterial blood = 7,4pH of the venous blood = 7,36

Decrease of pH is called acidosis. Increase of pH is called alkalosis.

Durable change of pH even on 0,1 - 0,2 may be lethal. The borders of pH changes, which are compatible with life, are 7,0 – 7,8. But these changes should not be durable, because the disturbance of pH level can lead to death.

Mechanisms which provide the constant pH level

Since pH of the blood is one of the most important home-ostatic factors, its maintenance on the constant level is pro-vided by many organs and systems of the organism.

25

The first “mean of protection” for the constant pH are buffer systems of blood. Every buffer system consists of two substances – weak acid and strong base. In process of metabo-lism formation of acid products is faster than basic products that are why the danger of acidosis in organism is higher. That is why in buffer pair acid-base, base capacity is higher and buf-fer systems are more resistible for the influence of acids. So, for the change of pH to the base side we should put 40 - 70 times more of NaOH, than to water, and to change pH to the acid side 300 - 350 times more of HCl.

There are 4 buffer systems of blood:1) hydrocarbonate;2) phosphate;3) hemoglobin;4) protein.

Hydrocarbonate buffer system consists of carbonic acid – H2CO3 and sodium hydrocarbonate – NaHCO3 in corre-lation 1:20. Principle of its functioning lies in: when acid is en-tering the blood (for example lactic acid, C3H6O3), which is stronger than carbonic acid, base reserve provides exchange of ions with formation of carbonic acid, which dissociates on CO2

and H2O.

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

OH ОН CO2 H2OLactic acid

This process is especially active in lungs, where CO2 is immediately loosed from the organism, which provides mainte-nance of pH on the constant level and prevents acidosis.

26

In case basic products enter the blood, the acid reservoir pro-vides exchange of ions with the formation of bicarbonate and water:

R+OH¯ + H+HCO3¯ RHCO3 + H2 O

RHCO3 is used to refill the buffer or released through kidneys. Usage of НСО3¯ leads to the leakage of CO2 and re-leasing of it through the lungs decreases.

So, hydrocarbonate buffer is the most mobile, powerful enough (its capacity is 13%), it is closely related with respira-tory system.

Phosphate buffer system consists of acid sodium salt of phophatic acid (NaH2PO4) and basic (Na2HPO4) in correlation 1:4. It is functioning by the same principle, like hydrocarbonate buffer. Due to low quantity of phosphates in blood, its capacity is low (5% of total capacity).

Hemoglobin buffer is represented by deoxidated hemo-globin (HHb) and potassium salt of oxidated hemoglobin (KHbO2) (75% of total capacity)

In capillaries of the tissues due to accumulation of acid metabolites there is danger of blood acidosis and hemoglobin is behaving like a basic substance:

KHbO2 → O2 + КHb (reaction of deoxygenation)

Hemoglobin, separated from oxygen has higher ability to bind H+ protons.

KHb + HR¯ → HHb + KR¯HHb + CO2 → HHbCO2

ННbCО2 is transported to the lungsHHbCO2 → CO2 + HHb

In lungs due to release of CO2 there is danger of blood alkalosis and hemoglobin behaves like an acid.

27

HHb:1) the source of protons: HHb → H+ + Hb¯;2) supports formation of H2CO3, which also dissociates with formation of protons:HHb + KHCO3 KHb + H2CO3

Н+ HCO3¯

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

COOH - the source of Н +

R NH2 - binds Н+

Buffer systems are not only in blood, but also in tissues, where they keep pH on the constant level. The main buffer sys-tems of the tissues are protein and phosphate buffer.

Buffer systems do not only reduce the pH shift, but they also predict total changes in pH level. That’s why for the effec-tive maintenance of acid-base balance, other organs and sys-tems are involved. For the fast compensation of pH lungs are involved due to its ability to regulate the amount of excreted CO2. The compensatory reactions of kidneys in form of de-crease of hydrocarbonate reabsorbtion, acidogenesis and amo-niogenesis, develop after 6-12 hours, or even days. Other or-gans also work to maintain of constant pH level. So, sweat-glands are able to excrete some products of metabolism (lactic acid), liver uses lactic acid of blood for glycogen biosynthesis, heart uses lactic acid like a substrate for oxidation.

28

Features of acid-base state of the blood1 pH = 7,36 – 7,4.2 Partial pressure of CO2 of the arterial blood (pCO2) = 35 –

45 mmHg.3 Actual bicarbonate of the blood (basic reserve of the blood)

– the true concentration of bicarbonate ion HCO3¯ in practi-

cal state of the arterial blood in the bloodstream. AB = 22 – 25 mmol/l4 Standard blood bicarbonate (SB) – the concentration of the

bicarbonate ion in full saturation of the hemoglobin with oxygen (shows the shift of pH, which is not associated with breathing). Normally SB = AB.

5 Buffer bases (BB) – the total amount of concentrations of all ions in the blood, which have buffer properties in full saturation of the blood with oxygen.BB = 40 – 48 mmol/l.

The special feature of it is that its value does not depend on changes of pCO2 that allows estimating the condition of acid-base balance of the organism independently from respi-ratory and not respiratory (metabolic) functions.

6 Buffer excess (BE) – the difference between the amount of buffer bases in patients organism and normal values.

HBO = -2,5 + 2,5

Age features of physical and chemical properties of the blood

Newborns and babies of the first year have different fea-tures of the blood than adults. So, newborns have higher den-sity and stickiness of the blood, which is determined by higher erythrocytes concentration. Till the end of the first month of life, these features decrease and approach to the one, adults have, or they become lower.

29

Placental blood circulation and labor complicate inter-change of gases. That is why children have acidosis before birth (pH = 7,13 – 7,23). During the first hours (or days) after birth, acidosis gradually disappears.

The concentration of plasma proteins in newborn organ-ism is lower (50 – 56 g/l). It will reach the level of adult in age of 3 – 4 years. The high concentration of γ-globulins is specific for the newborn, which the newborn gets from the mother. Till the end of third month its content decreases, but in future, with the help of its own antibodies formation, it will gradually in-crease. The concentration of α and γ-globulins reaches the adult level till the end of the first year of life.

With age, most of the physical and chemical properties of the blood (pH, osmotic pressure, sodium and potassium con-centration, viscosity), stay on the same level. Other features can change. So, ECR increases, osmotic resistance of erythro-cytes, hematocrit, ablolute and relative albumins concentration decrease.

Questions for self-control:

1 Functions of the blood.2 The volume of circulating blood (VCB). Factors, which de termine VCB.3 The composition of peripheral blood.4 Hematocrit. Factors, which determine hematocrit. Methods of hematocrit definition.5 The importance of water.6 The composition and functions of plasma proteins.7 The role of oncotic pressure in redistribution of water in organism.8 The importance of electrolytes.

30

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

Tests for self control:

1 The part of blood volume, which is occupied by erythrocytes:a) volume index;b) stickiness;c) erythrocyte sedimentation rate (ESR);d) color index;e) hematocrit;f) no correct answer.

2 Cations of blood plasma are:a) sodium;b) chlorine;c) bicarbonate;d) potassium;e) calcium;f) phosphate;

g) magnesium;h) sulphate.

31

3 Name the main function of blood plasma electrolytes:a) formation of hydrodynamic pressure;b) formation of hydrostatic pressure;c) formation of oncotic pressure;d) formation of osmotic pressure;e) formation of blood stickiness;f) protein transport;g) no correct answer.

4 The main role in the formation of oncotic pressure is:a) electrolytes;b) albumins;c) globulins;d) erythrocytes;e) leucocytes;f) no correct answer.

5 What are the functions of plasma proteins:a) participating hemostasis;b) protecting function;c) transport function;d) no correct answer?

6 Name the cases when hematocrit decreases:a) the increase of the erythrocytes amount in blood;b) the decrease of the erythrocytes amount in blood;c) the decrease of the blood volume;d) the increase of the blood volume;e) no correct answer.

7 The amount of what cells determines blood stickiness:a) monocytes;b) lymphocytes;c) thrombocytes;

32

d) eosinophils;e) basophiles;f) erythrocytes;g) neutrophils;h) no correct answer?

8 Name the components of hydrocarbonate buffer system:a) sodium hydrocarbonate;b) potassium hydrocarbonate;c) magnesium hydrocarbonate;d) carbonic acid;e) hydrochloric acid;f) no correct answer.

9 In normal state pH of the arterial blood is:a) 7,32;b) 7,36;c) 7,40;d) 7,44;e) no correct answer.

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

a) the increase of oncotic pressure of the blood in capillar-ies;b) the decrease of oncotic pressure of the blood in capillar-ies;c) the increase of oncotic pressure in the intercellular medium;d) the decrease of oncotic pressure in the intercellular medium;e) the increase of the hydrostatic pressure of the blood in capillaries;

33

f) the decrease of the hydrostatic pressure of the blood in capillaries;g) the increase of the hydrostatic pressure in the intercellu-lar medium;h) the decrease of the hydrostatic pressure in the intercellu-lar medium.

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

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

12 When pH of the blood is 7,32, it means:a) normal state;b) acidosis;c) alkalosis;d) hypovolemia;e) hypervolemia;f) no correct answer?

13 What happens to the cell in hypertonic solution:a) oedema;b) haemolysis;c) plasmolysis;d) immediate death;e) no correct answer?

14 What is the normal quantity of the blood:a) 1/5 of the body mass;b) 10 – 15% of the body mass;c) 6 – 8% of the body mass;

34

d) 4 – 5% of the body mass;e) no correct answer?

15 What are the main buffer systems of tissues:a) hemoglobin buffer;b) hydrocarbonate buffer;c) phosphate buffer;d) protein buffer;e) no correct answer?

35

CHAPTER 2 Physiologies of Erythrocytes

Definition of erythron

Erythron is the total mass of erythrocytes in the organism (the one circulating, the one that are stored, the one in organs of formation and degradation), and also the mechanisms of reg-ulation of their amount.

Functions of erythrocytes1 Respiratory function. Transport of the oxygen – is the

main function of erythrocytes, because this function in hu-man organism is performed only by them.

2 Transport function. Transport of CO2, proteins, hormones. 3 Buffer function. Maintaining of pH level in blood with the

help of hemoglobin buffer system.4 Maintaining the rheological properties of blood, namely

stickiness (increasing the amount of the erythrocytes leads to increasing the stickiness, when decreasing – decreasing the stickiness).

5 It makes the blood to belong to the blood group. There are aglutinogens on the erythrocyte membrane, which define the blood group.

6 Participate in the maintenance of the metabolism of salts and water. Erythrocytes can absorb the water on their sur-face, increasing the osmotic pressure or ions, decreasing the osmotic pressure.

7 Participate in homeostasis. Erythrocytes are the part of red clot; they are a matrix for the formation of protrombinase. Degraded erythrocytes co-operate in hypercoagulation and formation of clot.

Common functional characteristics of erythrocytes

36

I The amount of erythrocytesThere are 25•1012 erythrocytes in the blood of the human

organism. If we are to make the chain of all these erythrocytes, then its length is going to be 200 000 km. This chain can sur-round the Earth by equator 5 times.

The amount of erythrocytes in peripheral blood for male is 4 - 5•1012/liter, for female is 3,5 - 4,5•1012/liter.

The decrease in amount of erythrocytes is erythropenia or anemia. It can be absolute and relative.

Absolute erythropenia is the decrease in the total amount of erythrocytes in the organism. Its reasons:1) increase in the haemolysis of erythrocytes (due to exposure

to radiation, poisons, toxins, transfusion of the incompatible blood etc.);

2) loss of blood;3) decrease in speed or stopping of the erythropoiesis (because

of deficit of blood formation factors – iron, vitamins B6, B12, folic acid; because of erythropoietins deficiency when the kidneys are pathologic; depression of blood formation func-tion of red bone marrow);

Relative erythropenia – decrease in erythrocytes amount in blood volume unit when the blood is diluted. Its reasons: water retenssion in the in pathologies of the kidney; injecting the blood alternatives.

The increase of erythrocytes amount is erythrocytosis. It can be absolute and relative.

Absolute erythrocytosis is the increase of the erythrocytes amount in the organism. Its reason: increased of erythropoiesis because of the parcial pres-

sure in the air when in high altitude; because of great amount of erythropoietins during hy-

poxia among the patients with chronic sickness of the heart and lungs;

37

because of leucocytosis.Relative erythrocytosis is the increase in erythrocytes

amount in blood volume unit when the blood is concentrated. Its reasons: much of sweating, nausea, diarrhea; scorches; shock; cholera, dysentery; hard muscle work (because of erythrocytes leaving the

spleen blood storage).

Methods of counting the amount of erythrocytes1 Counting with the help of an autoanalyzer, based on erythro-

cytes electroconduction.2 Counting with celoscope, based on ability of erythrocytes to

absorb light.3 Counting with Burker’s camera with Goryaev’s net.

II Shape of erythrocytesErythrocyte has a shape of biconcave disc, which when

cut transversely, looks like dumb-bells. This shape helps ery-throcytes to fulfill their main respiratory function.

This shape provides:1) increase in the diffusion surface of erythrocyte.

With the help of this shape the internal surface of erythrocyte is 20% higher, than the one it can obtain when it is globe-shaped.

The total surface of all erythrocytes is 3800 m2; it is 1500 times larger, than the surface of human body.

2) shortening of diffusion distance. There is no point inside the erythrocyte, which will be more far than 0.85 mkm from the surface. If the erythrocyte is round shaped, its center would be in 2.5 mkm from the surface.Variations in shape of red blood cells

38

The following are the abnormal shape of red blood cells. Some of these abnormal shapes of the red blood cells occur in different types of anemia. Crenation: Shrinkage when in hypertonic solution. Spherocytosis: Globular form when in hypotonic solution. Elliptocytosis: Elliptical shape when in certain types of anemia. Sickle cell: Crescentic shape when in sickle cell anemia. Poikilocytosis: Unequal shapes due to deformed cell mem-

brane. The shape will be flask, hammer or any other unusual shape.

III Diameter of erythrocytesThe average erythrocyte diameter is 7.5 mkm. The allo-

cation of erythrocytes in healthy human fits to normal erythro-cyte allocation or Praice-Jones curve (fig. 2.1). number of

cells 120

Healthy100

80

60 Perniciose anemia

40

20

1 3 5 7 9 11 13 diameter, mkm

Figure 2.1 - Curve of Praice-Jones.In healthy human most of erythrocytes has diameter 7.5

mkm. There are also erythrocytes in blood of larger or smaller diameter in blood, but they are not much. If there is erythropo-

39

letic disorder then there are changes in Praice-Jones curve. In macrocytosis, there is increase in the number of erythrocytes, larger than 8 mkm (the diameter of some can reach 12 mkm) – the curve moves to the right. In microcytosis, there is the in-crease of the number of erythrocytes, smaller than 6 mkm (some can be even 2.2 mkm) – the curve moves to left. In per-nicios anemia there is poikilocytosis – the state when erythro-cytes of different shape are circulating in blood.Variations in size of red blood cells

The size of the red blood cells alters in various conditions. Mi-crocytes are the red blood cells of small size and are present in the following conditions: iron deficiency anemia; prolonged forced breathing and; increased osmotic pressure in blood.

Macrocytes are the red blood cells with larger size. The macro-cytes are present in the following conditions: megaloblastic anemia; muscular exercise and; decreased osmotic pressure in blood.

IV Erythrocyte is a non-nuclear cellThe loss of nucleus causes:

1) the increase of erythrocyte capacity (it is filled with hemo-globin);

2) the decrease of oxygen usage. Erythrocytes is used an amount of the oxygen, which is 200 times less than the cells that form erythrocyte, that have nucleus. The erythrocyte, supporting the whole body with oxygen, is using the less part of it;

3) the absence of nucleus and the presence of elastic mem-brane, allow erythrocyte to change its form easily to pass through thin capillaries.

40

V Plastic features of erythrocyteIt is the ability of the erythrocyte to change its shape.

Due to plastic features erythrocyte can pass through the capil-laries, which is twice thinner than erythrocyte itself. The plastic features are provided by protein spectrin, which is situated in the membrane and inside the erythrocyte itself. Spectrin is 75% of all the proteins of erythrocyte. Its functions are:1) it forms the cytoskeleton and keeps the shape of erythro-

cytes;2) it gives membrane elastic features. Due to ability to contract

it helps erythrocytes to change their form.

VI Osmotic resistance of erythrocytesIt is the ability of erythrocyte membrane to resist osmotic

haemolysis.The osmotic pressure inside the erythrocyte is a little

higher than osmotic pressure of blood plasma. That’s why wa-ter is entering the erythrocyte, what provides normal turgor of the cell. In hypotonic solution, water moves inside the erythro-cyte, resulting in swelling and rupture of the cell. It is called osmotic haemolysis.

The measure of osmotic resistance is the concentration of hypotonic solution, which causes haemolysis. In normal state in human organism the destruction of the erythrocytes with the smallest resistance begins in 0,54% solution of NaCl. This value is called minimal osmotic resistance. When concentration of NaCl is 0,42%, – 50% of erythrocytes are destructed, when it is 0,34% – all erythrocytes are destructed. This value is called maximal osmotic resistance (fig. 2.2).

During sickness (for example anemia) osmotic resistance is decreased and haemolysis occurs in higher concentrations of NaCl.

41

Figure 2.2 - Osmotic resistance of erythrocytes

VII Lifespan and fate of red blood cellsAverage lifespan of red blood cell is about 120 days. The se-

nile red blood cells are destroyed in reticuloendothelial system. When the cells become older (120 days), the cell membrane be-comes 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 capil-laries of spleen because the splenic capillaries have a thin lumen. So, the spleen is usually called ' graveyard of red blood cells. The destroyed red blood cejls are"fragmented. From the fragmented parts, the hemoglobin is released. The iron and globin parts of the hemoglobin are separated with the production of bilirubin. Iron combines with the protein-apoferritin to form ferritin, which is stored in body. Globin also enters the protein depot. The bilirubin is excreted by liver through bile. Daily 10% red blood cells, which are senile, get destroyed in normal young healthy adults. This causes release of about 0.6 g% of hemoglobin into the plasma. From this 0.9 to 1.5 mg% bilirubin is formed.

42

Determination of lifespan of red blood cellThe lifespan of the red blood cell is determined by radioiso-

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

Erythrocyte sedimentation rate (ESR)Erythrocytes can not sedimentate inside blood vessels.

Due to: constant blood movement; the charge of blood vessel wall and the charge of ery-

throcyte is the same (negative) so cells repel from it.If we put the blood inside the test-tube and add anticoag-

ulant than in few minutes we can observe the sedimentation of erythrocytes, because the density of erythrocytes (1,090 g/cm3) is higher, than the density of blood plasma (1,025 – 1,034 g/cm3). The mechanism of the process is as follows. At first ery-throcytes form complexes with each other (10-12 erythrocytes form “monetary column”). After these complexes interact with plasma proteins, they become heavier and they start to settle faster. Due to this process is not equable in time (slow in the beginning, faster in the end) ESR is determined for the fixed period of time, usually 1 hour.

In normal state ESR of men is 2-10 mm/hour, of women 2-15 mm/hour.

Factors, which affect ESRThe main mechanism of influence of all factors is the

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

43

The first group of factors – plasma factors:1 The protein content of blood plasma

The influence of this factor is proved in the following ex-periment. Erythrocytes of the patient with increased ESR are put in blood plasma of the healthy man with the same blood group. Erythrocytes of the patient sediments with normal speed, otherwise erythrocytes of healthy man sediments in pa-tient’s blood plasma with higher speed.

Different proteins affect ESR in different ways. When al-bumins concentration is increased, ESR decreases. When con-centration of high molecular proteins, globulins or fibrinogen increases – ESR increases. Possibly, high molecular proteins decrease electric charge on the erythrocytes membrane, depress the electric repulsion of blood cells. Due to this the aggregation properties of erythrocytes increase, ESR increase. Globulins concentration increases in case of inflammatory processes, in-fectious sicknesses and malignant tumors. That is why these patients have increased level of ESR.

The amount of fibrinogen increases in 2 times in the sec-ond half of pregnancy, that’s why before the delivery ESR of the pregnant woman can reach 40 – 50 mm/hour.

2 Plasma volumeWhen increased plasma volume, hematocrit decreases,

blood stickiness decreases, and as a consequence ESR in-creases.The second group of factors – erythrocyte factors.1 The amount of erythrocytes in blood volume (hemat-

ocrit)The higher amount of erythrocytes – the higher stickness

– the lower ESR.The lower amount of erythrocytes – the lower stickness –

the higher ESR.This is the reason of increase in ESR in anemic patients.

2 The ability of erythrocytes to aggregate

44

The increase of erythrocyte ability to aggregate leads to the decrease of stickiness, because the resistance of the aggre-gates to friction is lower, than the resistance of separate cells because of the decrease of correlation of the surface to the vol-ume. Aggregates sediments faster and ESR increases. The in-crease of erythrocyte ability to aggregate is observed when in-flammatory processes and malignant tumors.3 Erythrocytes shape

The change of the erythrocytes shape (for example when sickle-cell anemia) or its modification (for example, when per-nicious anemia) can cause the oppression of the erythrocytes ability to aggregate. It causes the increase of stickiness and, as a consequence, the decrease of ESR.

Except these factors, there are some other ones, which af-fect ESR. For example, steroid hormones (estrogen, glucocor-ticoid hormones) and some medicine (salicylates) increase ESR. Erythrocytes sedimentation rate increases when the con-tent of cholesterol in blood increases, during alkalosis, and it decreases when content of bilious pigments and bilious acids in blood increases and also during acidosis.

Functional properties of erythrocyte componentsComponents of erythrocyte are: membrane; fermentative systems; hemoglobin (Hb).Erythrocyte membrane consists of bilipid layer, which

is bound with proteins. Membrane has plaques, which are filled with glycoproteins and other proteins, carboxyl groups, which form negative charge on the erythrocyte membrane, which is called zeta-potential. The thickness of the membrane is 10 mkm. It is 1 million times more permeable for anions than for cations. Anions of HCO3

-, Cl-, and O2, CO2, H+ and OH- are easily passing through it; Na+ and K+ are not passing.

45

On the external side of the membrane there are sialic acids and glycoproteins, which have antigenic properties and define the blood group.

On the internal side of the membrane there are glycolytic enzymes, Na-K-ATP-ase, glycoproteins, hemoglobin.

Fermentative systems of erythrocytes are represented by:

fermentative system of glycolysis; fermentative system of pentose cycle; glutationperoxydase fermentative system.

Metabolism of erythrocytes is different from other cells metabolism.

At first, erythrocyte is using less than any other cell. That’s why the amount of ATP formed is small. Mitochondria is absent in erythrocyte and ATP is formed in glycolysis.

At second, metabolism is directed to maintain its ability to bind oxygen, the recovery of iron ion in heme structure is needed.

In the result of spontaneous oxidation of bivalent iron Fe2+ is transformed into trivalent iron Fe3+. And to bind the oxygen Fe3+ should be recovered to Fe2+ with the help of NAD•H+.

Fermentative system of glycolysis supports erythrocyte with:

adenosintriphosphate (ATP); recovered NAD•H+, which is used for recovery of Fe3+

into Fe2+; 2,3-biphosphoglycerate (2,3-BPG), is an important intra-

cellular regulator of hemoglobin functions.2,3-BPG regulates the affinity of hemoglobin to the oxygen.2,3-BPG connected to hemoglobin helps its deoxygenation.

Fermentative system of pentose cycle supports erythro-cyte with NADP•H+, which is the component of antioxidative system and needed for the recovery of glutation.

46

Glutationperoxidase system – is antioxidative system, which protects a number of erythrocyte enzymes, which have SH-group, from oxidation.

Hemoglobin (Hb) – is the main erythrocyte compound. It makes 90% of the total solids of the cell. The fact that hemo-globin is kept inside the cell is very important. If hemoglobin was inside the blood plasma, it could cause a number of disor-ders.1 A large amount of free Hb does a toxic influence on differ-

ent tissues (neurons, kidneys).2 In bloodstream Hb is turned to methemoglobin, but in the

erythrocyte there are fermentative systems, which predict this to happen.

3 The amount of hemoglobin, needed for the transport of the enough amount of oxygen will increase stickiness.

4 Hb will increase an oncotic pressure of plasma that will lead to dehydration of tissues.

5 The part of Hb will be filtrated through the kidneys and it will choke pores of kidney’s filter.

The structure of hemoglobinHemoglobin (Hb) – is a red pigment, chromoprotein,

which is situated in erythrocytes and transports oxygen.There is 900 g of hemoglobin in human with body weight

70 kg. There are nearly 400 millions of hemoglobin molecules in one erythrocyte. The molecular mass of hemoglobin is 64 450.

Hemoglobin has globular molecule, which is formed with 4 subunits. Each subunit contains heme. Heme – is Fe-inclu-sive substance, the derivative of porphyrin. Heme molecule consists of 4 pyrrol. The ion of Fe2+ is situated in the center (fig. 2.3).

47

Deoxygenated hemoglobin Oxygenated hemoglobin

Figure 2.3 – Types of hemoglobin

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

There are 2α and 2β

The main hemoglobin forms and compositionsDepending of protein chains there are following forms of

hemoglobin in normal state. Hb P (primitive) in embryo for the first 7-12 weeks Hb F (fetal) in fetus. Appears on ninth week. Consists of

2α- and 2γ- chains. Hb F can bind and transport oxygen

48

Figure 2.4 – The schematic image of hemoglobin A

easier (due to lesser similarity HbF to 2,3-BPG). That’s why in the blood of fetus there is enough amount of HbO2 formed, regardless lower tension of O2. Normally after birth fetal hemoglobin is changed to adult hemoglo-bin.

HbA1 (adult). It contains 2α- and 2β- chains. HbA1 is 95% of all hemoglobin of adult.

HbA2 – it contains 2α and 2δ chains. It is 5% of all he-moglobin of adult.In some inheritable diseases, there are defects of genes,

which encode α- or β- chains and the synthesis of Hb is dis-turbed. These sicknesses are called thalassemias.

In α-thalassemia, the synthesis of α-chains is disturbed. Erythrocytes are target-shaped, that’s why α-thalassemia is also called target-shaped anemia. In β-thalassemias synthesis of β-chains is disturbed (Kulee sickness).

Defects of the primary structure of hemoglobin also be-long to the pathological changes of hemoglobin. Mutative genes, which produce abnormal hemoglobins, are widely spread. There are a lot of forms of abnormal hemoglobins. For example, if glutamate is changed to valine in β-chain, patho-logical HbS is formed. In deoxygenated state its dissolubility decreases 100 times, and it forms sediment. These crystals de-form erythrocyte. Erythrocyte gets sickle-shape, hardly passes through small capillaries and phagocyted by macrophages. It is called sickle-cell anemia.

Main physiological compositions of hemoglobin1) HbO2 (oxygemoglobin) is the composition of Hb with

oxygen. It has red color, which define the red color of the arte-rial blood.

Due to no oxidation occuring during interaction between Hb and O2 and oxidation degree of iron does not change; the reaction is called oxygenation (not oxidation).

49

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

3) HbCO2 (carbhemoglobin) – the composition of Hb with CO2.

Pathological composition of Hb:1) HbCO (carboxyhemoglobin) – the composition of Hb with CO.

Chemical relativity of Hb to CO is in 300 times higher than O2. That’s why carbon monoxide displaces O2 from hemo-globin, decreasing the ability of the blood to bind oxygen. Even small number of CO leads to the significant increase in forma-tion of HbCO. When concentration of the CO in the air is 0,1% - 80% Hb binds not with O2, but with CO. When concentration of CO in the air is 1%, in few seconds it will cause death.

It is dangerous because HbCO is persistent and Hb can-not transport oxygen anymore.

Low intoxication with CO is a reversible process and af-ter breathing fresh air, CO will gradually detach. Breathing with clean oxygen has positive effect.

Normally HbCO is 1% of all the Hb. In smokers body it is 3%, after heavy pull – 10%.2) Met Hb (HbOH - methemoglobin) – hemoglobin, which contains Fe3+ and has brown color. Oxidation of Fe2+ to Fe3+ in hemoglobin occurs when interacting with strong oxidizers (KMnO4, aniline), and also with medicine of oxidative proper-ties. Insignificant oxidation of hemoglobin to methemoglobin also occurs in normal conditions. But with the help of ferment-ing systems of erythrocyte (NADH-methemoglobinreductase system) methemoglobin turns in to hemoglobin. Inherited ab-sence of this fermentation can cause inherited methe-moglobinemia.

50

In pathological conditions, when methemoglobin is formed, blood with high oxygen content circulates in the or-ganism, but it is not entering tissues.

The amount of hemoglobin in blood of healthy human is 140-160 g/L for men, 120-140 g/L for women, 200 g/L – for newborns.

The definition of hemoglobin content:1 Definition of the amount of bonded oxygen (1g of Hb can

bind 1,34 ml of O2).2 Analysis of iron level in blood (iron content in hemoglobin

is 0,34%).3 Tintometry (comparison of blood color with color of stan-

dard solution) – Sali method.4 Spectrophotometry.

Method 1 and 2 require sophisticated apparatus. Third – is inaccurate. Fourth method is very popular nowadays. Blood is mixed with the solution of potassium ferricyanide, potassium cyanide, sodium bicarbonate. These substances will cause de-struction of erythrocytes; Hb turns to cyan methemoglobin (HbCN). Unlike Hb, HbCN is stable and it can be stored for few weeks. The solution is rayed with monochromatic light with λ = 546nm, then extinction is defined. The content of Hb is defined by special calibration scale.

T he following showings are important in estimation of eryt forms of anemia:

1) Average hemoglobin content in one erythrocyte (AHC) – characterizes the absolute number of Hb in the erythrocyte.

51

AHC = Hb / E

Normally AHC = 26-36 picogramWhen AHC is normal, than erythrocytes, they are called

normochromic.When AHC is less than normal, erythrocytes are called

hypochromic.When AHC is higher than normal, erythrocytes are called

hyperchromic.2) Color index (CI) – the index which characterizes the rela-

tive content of Hb in 1 erythrocyte.CI = Hb / first three numbers in the amount of erythrocytesNormally CI = 0,85 – 1,15

If CI is normal, erythrocytes (and anemia) are nor-mochromic. If CI is lower than normal – hypochromic. If CI is higher than normal – hyperchromic.3) Oxygen-carrying capacity of blood (OCC) – the amount

of oxygen, which is transported with 1 liter of blood1 g of Hb can bind 1,34 ml of O2 - it is Hufner's number.

OCC = Hufner’s number • Hb (in g/l).

Regulation of erythrocyte content in peripheral bloodThe erythrocyte content in peripheral blood of the adult is

3,5 – 5•1012/liter. The changes of this value (decrease or in-crease) can lead to the dangerous changes in human organism. The decrease in the erythrocyte amount leads to the interrup-tion of the oxygen transport in blood, which cause ischemia of organs and tissues. The increase in the erythrocyte amount is the reason of the increase in blood stickiness and the increase of load on heart. When there is essential increase of blood stickiness, the movement of the blood in vessels is impossible.Regulation of erythrocyte content is provided by regulation of its formation (erythropoiesis) and destruction (haemolysis).

52

Variations in number of red blood cells

Physiological variationsI Increase in the red blood cell count is known as poly-cythemia. If it occurs in physiological conditions, it is called physiological polycythemia. It occurs in the following condi-tions:1) AgeAt birth, the red blood cell count is 8 -10 millions/cu mm of

blood, The count decreases within 10 days after birth due to destruction of cells causing physiological jaundice in some infant. However, in infants and growing children, the cell count is at a level higher than the value in adults.

2) SexBefore puberty and after menopause in females the red blood

cell count is similar to that in males. During reproductive pe-riod 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 kid-neys. 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 ex-ercise. This is because of mild hypoxia and contraction of spleen, which is the reservoir of blood.5) Emotional ConditionsThe red blood cell count is increased during the emotional condi-

tions like anxiety, because of sympathetic stimulation.6) Increased Environmental Temperature

53

The increase in the atmospheric temperature increases red blood cell count.

7) After MealsThere is a slight increase in the red blood cell count after taking

meals.II Decrease in red blood cell count occurs in the follow-

ing physiological conditions:1) High Barometric Pressures

At high barometric pressures as in deep sea, when the oxy-gen tension of blood is higher, the red blood cell count decreases.2) After Sleep

The red blood cell count decreases slightly after sleep.3) Pregnancy

In pregnancy, the red blood cell count decreases. This is because of increase in extracellular fluid volume. Increase in ex-tracellular fluid volume, increases the plasma volume also resulting in hemodilution. So, there is a relative reduction in the red blood cell count.

Mechanisms of erythrocytes formationErythrocytes are formed in tissues of blood formation: in

the yolk sac of the embryo, in liver and spleen of the fetus and in the red bone marrow of adult.

There are polypotent stem cells, which form all blood cells.

There are 6 classes of red bone marrow cells:1st class – stem cells (polypotent).2nd class – half-stem cells (partially determined mediators of hemopoesis).3rd class – erythropoietin-sensitive cells (unipotent mediators of hemopoesis). Cells of these classes are morphologically the same.4th class – blasts – erythroblast.

54

5th class – maturating cells (cells, which are differentiating) – normoblast.Two types of processes occur in maturating cells:

Cells lose their organelles (nuclei, mitochondrias, and endoplasmic reticulum)

Synthesis and accumulation of hemoglobin occur.Proerythroblast has large nucleus, it is characterized by

intensive proliferation (it divides every 8-12 hours).Basophilic erythroblast – has smaller size, dyes with ba-

sic stains. Hemoglobin appears in these cells.Polychromatophilic erythroblast – has size, smaller than

basophilic, dyes with both basic and acidic stains. Accumula-tion of hemoglobin occurs.

Oxyphilic erythroblast divides on the initial stage. Later destruction of nuclei occurs, then nuclei disappear and cells stop their division. They dye with acidic stains. The amount of Hb increases.6th class – mature cells (already differentiated) reticulocyte, erythrocyte.

The amount of reticulocyte in the blood testifies about the intensity of erythropoiesis. Normally its amount is 1% out of all erythrocytes. The amount of reticulocytes increases when activation of erythropoiesis. But in any case erythropoiesis can be only 5-7 times more intensive comparably to the normal level.

Due to absence of large erythrocytes depot in the organ-ism, liquidation of anemia after blood loss occurs with the help of erythropoiesis. But the intensity of erythropoiesis in red bone marrow starts after 3-5 days, and in peripheral blood it is noticeable after 2-3 weeks.Next factors of blood formation those are required for erythro-poiesis: Iron (for the synthesis of heme). The daily need of iron is

20-25 mg. 95% of this amount, organism gets from the he-

55

moglobin of destroyed erythrocytes and 5% (1mg) – from food.

Vitamin B12 – the external factor of blood formation. Or-ganism gets it from food, but it is absorbed only in pres-ence of internal factor of blood formation – Castle’s factor, which is secreted by glands of the stomach.

Folic acid. Gets into the organism from vegetative food.Vitamin B12 and folic acid are required for the synthesis of nucleic acids and globin.

Vitamin C – participates in the metabolism of iron. Needed in formation of heme, increases the activity of folic acid.

Vitamin B6 – formation of heme. Vitamin B2 – formation of lipid part of the erythrocytes. Pantothenic acid – synthesis of phospholipids of the ery-

throcytes membrane.

Mechanisms of the erythropoiesis regulation1 Nervous:

sympathetic nervous system stimulates erythropoiesis; parasympathetic – inhibits.

2 Humoral:Somatotropic hormone, adrenocorticotropic hormone,

catecholamines, hormones of thyroid gland (thyroxin) and male hormones stimulate erythropoiesis, female hormones – inhibit.

These nervous and humoral mechanisms are important for blood formation. They work not directly but with the help of specific mediators – “hormones of blood formation”.

Erythropoietin is a specific humoral stimulator of ery-thropoiesis. 80% of erythropoietins are produced by kidneys. Experimentally after nephrectomy the level of erythropoietins will decrease. Synthesis of erythropoietins will be inhibited in case of kidneys insufficiency. 20% of erythropoietins are pro-duced by macrophages. Hypoxia of kidneys is a stimulator of erythropoietin production. There is a protein, which can bind

56

oxygen in the perivestibular cells of kidneys. During sufficient oxygenation oxyform of hemoprotein blocks gene, which is re-sponsible for erythropoietin synthesis and erythropoietin is not produced. During hypoxia desoxyform (without oxygen) of hemoprotein is produced, which cannot block this gene and synthesis of erythropoietin occurs. Besides, when there is oxy-gen insufficiency a number of enzymes that are sensitive to hy-poxia are activated in kidneys. Phospholipase A2, which sup-ports the formation of prostaglandins, like prostaglandins E1

and E2, which activate adenylate cyclase and cause the increase of cAMP concentration that increases synthesis and secretion of erythropoietins.

Mechanisms of erythrocytes degradationMature erythrocytes circulate 100 - 120 days in the

bloodstream. After that they are engulfed by the cells of reticu-loendothelial system of the bone marrow, macrophages of the liver and spleen. Small amount can go through haemolysis in the bloodstream.

The main site of the erythrocyte degradation is the spleen. Not only spleen, but any other tissue can degrade ery-throcytes, prove of this – is the fact that extravasation gradually disappear in any part of the human body.

The reason of erythrocyte degradation (haemolysis) is ag-ing. Due to aging, the rate of metabolic processes decreases: the activity of the enzymes of glycolysis and pentose phosphate pathway decrease, as the consequence the number of ATP, NAD•H and other important substances will decrease. The con-sequences of decrease of the rate of metabolic processes are:1) elasticity loss: erythrocyte becomes unable to pass through

narrow site of the bloodstream and dwells there. One of these sites is spleen, where the distance between trabecula-tions is less than 3 mkm. Erythrocytes dwell here, the part of the cells and hemoglobin are engulfed by macrophages;

57

2) the decrease in the ability to transform Fe3+ into Fe2+ (due to insufficiency of NAD•H), which causes the disturbance of gas transport function;

3) appearance of the sialic acids on the erythrocyte surface. Macrophages have receptors to this groups that helps its co-operation and degradation.

Engulfed erythrocytes go through haemolysis in the cells of mononuclear phagocytic system (macrophages of spleen, red bone marrow, Kupfer cells in liver). Hemoglobin is degraded to heme and globin. Biliverdin is formed from heme. Biliverdin forms bilirubin, which is called non-conjugated (it is insoluble in water and reacts with diasoreagent only after treatment with alcohol) and it is transported in the liver, where it interacts with glucuronic acid. This bilirubin is called conjugated (it is solu-ble in water and it reacts with diasoreagent). Conjugated biliru-bin and very small amount of non-conjugated is released with bile into small intestine, where with the help of enzymes of small intestine microflora, it turns into urobilinogen. There are two possible ways of further transformation. The large part of urobilinogen in the large intestine forms stercobilinogen, which is excreted with faeces or with urine after absorption into the bloodstream in the site of the superior and medium hemorrhoid plexus of rectum. The lesser part of the urobilinogen partici-pates in hepato-intestinal circulation – absorbed in the small in-testine, gets to the liver, partially oxidized and partially return to the intestine through bile ducts.

Forms of haemolysisThe destruction of erythrocyte membrane accompanied

with the release of the hemoglobin into the blood plasma is called haemolysis. Heamolyzed blood becomes transparent.Depending on the reasons of degradation, there are other next types of haemolysis:

58

1 Mechanical haemolysis. It is caused by the mechanical degradation of the erythrocyte membrane. For example due to ruin of the erythrocytes in the vessels of foot; or due to shaking of glass with blood.

2 Osmotic haemolysis. It occurs when the osmotic pres-sure inside the erythrocyte is higher than in blood plasma. In this case, water due to laws of osmosis enters the erythrocyte, its volume increases and the degradation of membrane occurs. Reasons of osmotic haemolysis are:

The decrease of the osmotic pressure of the medium, where the erythrocyte is (hypotonic solution);

The increase of the osmotic pressure in the erythrocyte itself due to increase of the membrane permeability or disturbance in work of Na-K pump.

3 Chemical haemolysis. It is haemolysis, which occur un-der the influence of the substances that can degrade erythrocyte membrane (ether, chloroform, alcohol, bilious acids, saponine and others).

4 Thermal haemolysis. It is haemolysis, which is caused by the influence of high or low temperatures. For example dur-ing deep-freeze of the blood.

5 Biological haemolysis. It is haemolysis, which develops after transfusion of incompatible blood and after sting of some snakes.

Questions for self-control1 The definition of erythron.2 Functions of erythrocytes.3 The amount of erythrocytes. The definition of erythrocytosis

and erythropenia.4 Methods of calculating erythrocytes.5 The shape of erythrocytes.6 Diameter of erythrocytes. Praice-Jones curve.7 Plasticity of erythrocytes.

59

8 Osmotic resistance of erythrocytes.9 Erythrocyte sedimentation rate (ESR). Factors, which affect

ESR.10 Functional properties of erythrocyte elements.11 Forms and compositions of hemoglobin.12 Methods of determination of hemoglobin content in periph-

eral blood.13 Values, which are used for erythropoiesis diagnostics.14 The formation of erythrocytes in the organism.15 Mechanisms of erythropoiesis regulation.16 Reasons and mechanisms of erythrocytes degradation.17 Forms of haemolysis.

Tests for self-control1 Which value shows relative content of hemoglobin in every single erythrocyte:

a) erythrocyte sedimentation rate (ESR) ;b) hematocrit;c) diameter of erythrocytes;d) Praice-Jones curve;e) color index;f) no correct answer?

2 Reticulocytes are immature forms of:a) erythrocytes;b) lymphocytes;c) neutrophils;d) monocytes;e) thrombocytes;f) basophils;g) eosinophils;h) no correct answer.

3 Name one organ where erythrocytes go through physiologi-cal degradation:

60

a) red bone marrow;b) lymphatic nodules;c) liver;d) spleen;e) lungs;f) kidneys;g) thymus;h) no correct answer.

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

5 What type of haemolysis will cause hypotonic solutions:a) chemical;b) biological;c) osmotic;d) thermal;e) no correct answer?

6 What is the normal value for erythrocyte sedimentation rate in male:

a) 0 – 1%;b) 1 – 10%;c) 2 – 10 mm/hour;d) 2 – 15 mm/hour;e) 7 – 15 mm/hour;f) no correct answer?

7 What cause the increase in ESR:a) increase in the number of erythrocytes;

61

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

8 How long do erythrocytes circulate in the bloodstream:a) 2 – 3 months;b) 5 – 6 months;c) 100 – 120 days;d) 40 – 50 days;e) 1 year;f) no correct answer?

9 What are the factors, which increase erythropoiesis:a) androgens;b) estrogens;c) somatotropic hormone;d) adrenalin;e) thyroxin;f) no correct answer?

10 What is the main function of erythrocytes:a) respiratory;b) protection;c) immune;d) trophic;e) energetic;f) no correct answer?

11 What is oxygen-carrying capacity of the patient, if his he-moglobin level is 100 g/l:

a) 100 ml;b) 125 ml;

62

c) 134 ml;d) 146 ml;e) no correct answer?

12 What are non-nuclear cells of the blood:a) lymphocytes;b) monocytes;c) basophiles;d) neutrophils;e) erythrocytes;f) no correct answer?

13 If concentration of 2,3-DPG will increase, what will happen to the amount of the oxygen, transported with hemoglobin:

a) decrease;b) increase;c) will not change?

14 What is the composition of hemoglobin with carbon diox-ide:

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

15 How many hemes are in one molecule of hemoglobin:a) 1;b) 2;c) 3;d) 4;e) 8?

63

CHAPTER 3 Blood groups

The definition of agglutinogens,agglutinins, agglutination

Agglutinogens (hemagglutinogens) – specific antigens, which are situated on the erythrocyte membrane. By chemical nature they are glycoproteins or glycolipids. There is an indi-vidual set of specific erythrocyte agglutinogens. Approxi-mately 400 are discovered now. Thirty are mostly common and they can be a reason of reactions after blood transfusion.

Agglutinins (isohemagglutinins) – specific antibodies, which are dissolved in blood plasma, they are related to γ-glob-ulins fraction.

Agglutination – is agglutination of erythrocytes, which occur in the result of reaction of antigen-antibody. As a rule, agglutination is accompanied by erythrocyte haemolysis. Ag-glutination occurs after similar agglutinogens meet agglutinins. Agglutinin has 2 active centers, which are why it can bind 2 erythrocytes by forming a “bridge” between them.

In 1901 austrian K. Landsteiner and in 1903 czech Y. Yanskiy found agglutinogens A and B on the surface of the erythrocytes and explained the agglutination.

Definition of the blood group by ABO system is based on the presence of aggluttinogens A and B on the surface of the erythrocytes. Agglutinins α and β are formed during the first year of life. Agglutinins are formed to agglutinogens which are absent on the erythrocyte surface (if erythrocytes have agglu-tinogen A, agglutinin β is formed, if B – α). Agglutinins relate mostly to immunoglobulins M (IgM). They are high-molecular immunoglobulins. IgM are typical hemolysins (when they in-teract with relative antigens on the erythrocyte membrane, they form substances, which destroy erythrocytes).

If similar agglutinogens and agglutinins meet: A with α,

64

B with β – agglutination occurs, which ends with haemolysis of erythrocytes. Lysis of erythrocytes performs with the help of complement system and proteolytic enzymes. Accumulation of destroyed erythrocytes leads to obstruction of capillaries and other complications, which can cause death. That’s why in nat-ural conditions human organism cannot have antigens and anti-bodies, which are relatied to each other, because it could lead to agglutination of own erythrocytes.

There are 4 blood groups in ABO system:

Blood group

Agglutinogens (erythrocytes)

Agglutinins (plasma)

І (О) – α, βІІ (А) А βІІІ (В) В α

ІV (АВ) А В –

Current ideas about ABO blood groupsTwo particular types of antigens are much more likely

than the others to cause blood transfusion reactions. They are the O-A-B system of antigens and the Rh system.

Four major O-A-B blood types, as shown in table above, depending on the presence or absence of the two agglutino-gens, the A and B agglutinogens.When neither A nor B agglu-tinogen is present, the blood is type O. When only type A ag-glutinogen is present, the blood is type A.When only type B ag-glutinogen is present, the blood is type B.When both A and B agglutinogens are present, the blood is type AB. These combi-nations of genes are known as the genotypes, and each person is one of the six genotypes. When type A agglutinogen is not present in a person’s red blood cells, antibodies known as anti-A agglutinins develop in the plasma. Also, when type B agglu-tinogen is not present in the red blood cells, antibodies known

65

as anti-B agglutinins develop in the plasma. Thus, note that type O blood, lthough containing no agglutinogens, does con-tain both anti-A and anti-B agglutinins; type A blood contains type A agglutinogens and anti-B agglutinins; type B blood contains type B agglutinogens and anti-A agglutinins. Finally, type AB blood contains both A and B agglutinogens but no ag-glutinins.

Titre of the Agglutinins at Different Ages

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

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

66

Relative rates of red blood cell production in the bone marrow of different bones at different ages.

Origin of Agglutinins in the Plasma

The agglutinins are gamma globulins, as are almost all antibodies, and they are produced by the same bone marrow and lymph gland cells that produce antibodies to any other anti-gens. Most of them are IgM and IgG immunoglobulin mole-cules.

But why are these agglutinins produced in people who do not have the respective agglutinogens in their red blood cells? The answer to this is that small amounts of type A and B anti-gens enter the body through food, through bacteria, and other ways, and these substances initiate the development of the anti-A and anti-B agglutinins. For instance, infusion of group A antigen into a recipient having a non-A blood type causes a typical immune response with formation of greater quantities of anti-A agglutinins than ever.Also, the neonate has few, if any, agglutinins, showing that agglutinin formation occurs al-most entirely after birth.

Agglutination Process In Transfusion Reactions

When blood is mismatched so that anti-A or anti-B plasma agglutinins are mixed with red blood cells that contain A or B agglutinogens, respectively, the red cells agglutinate as a result of the agglutinins’ attaching themselves to the red blood cells (Fig. 3.2). Because the agglutinins have two binding sites (IgG type) or 10 binding sites (IgM type), a single agglu-tinin can attach to two or more red blood cells at the same time, thereby causing the cells to be bound together by the agglu-tinin. This causes the cells to clump, which is the process of “agglutination.” Then these clumps plug small blood vessels

67

throughout the circulatory system. During ensuing hours to days, either physical distortion of the cells or attack by phago-cytic white blood cells destroys the membranes of the aggluti-nated cells, releasing hemoglobin into the plasma, which is called “hemolysis” of the red blood cells.

Figure 3.2 Agglutination reaction. People with type A blood have type A antigens on their red blood cells and antibodies in their plasma against the type B antigen. People with type B blood have type B antigens on their red blood cells and antibodies in their plasma against the type A antigen. Therefore, if red blood cells from one blood type are mixed with antibodies from the plasma of the other blood type, an agglutination reac-tion occurs. In this reaction, red blood cells stick together because of anti-gen-antibody binding. Acute hemolysis occurs in some transfusion reac-tions.

68

Antigens on red blood cells

Antibodies in plasma

Agglutina-tion reaction

Type Type

Sometimes, when recipient and donor is blood is mis-matched, immediate hemolysis of red cells occurs in the circu-lating blood. In this case, the antibodies cause lysis of the red blood cells by activating the complement system, which re-leases proteolytic enzymes (the lytic complex) that rupture the cell membranes. Immediate intravascular hemolysis is far less common than agglutination followed by delayed hemolysis, be-cause not only does there have to be a high titre of antibodies for lysis to occur, but also a different type of antibody seems to be required, mainly the IgM antibodies; these antibodies are called hemolysins.

Human’s blood group is defined by the antigen properties of erythrocytes. Antigens A and B are glycoproteins, which consist 75% of carbohydrates, 15% of amino acids and 10% of phospholipids. Antigen properties depend on the nature of the sugar in glycoproteins, in other words peculiarity of antigen is defined by its carbohydrate part.

Patients with O-group (I) have antigen H, which has three carbohydrate tails (N-acetylgalactosamine, galactose, fu-cose).

Erythrocytes of II group have fourth tail, connected to previous - N-acetylgalactosamin.

Erythrocytes of III group have fourth tail, connected to previous - galactose.

In erythrocytes of IV group part of glycoproteins ends with galactose and part ends with N-acetylgalactosamin (fig. 3.3).

There are two sites in genome of the cell, which is re-sponsible for blood formation – H and ABO, which are respon-sible for the synthesis of gene.

69

Figure 3.3 - Structure of glycoprotein of the erythrocyte membrane

In H-site one antigen is formed: H-gene, which encodes enzyme fucosyl-transferase that transports fucose to galactose and controls synthesis of antigen H.

In ABO-site three antigens are formed: Gene-O, which encodes protein that does not have fermenta-tion activity. A-gene, which encodes enzyme A specific transferase that transports N-acetylgalactosamine to fucose, subsequently anti-gen A is formed on the erythrocyte membrane.

70

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

Definition of ABO blood group system

The same principle is used to define the blood group in every system: to provide conditions for the agglutination of erythrocytes in the medium of standard isohemagglutinating serums or coliclones, which have high titre of antibodies for the examined erythrocytes antigens.

Standard serum is a prepared blood plasma of donors with different blood groups, without fibrinogen and has high concentration of antibodies for one or several antigens of one blood group.

There are 4 groups of serums in ABO system. The serum of group I has agglutinins α and β (colorless); group II – agglu-tinins β (light blue color); group III – agglutinins α (pink color); IV group – does not have agglutinins (yellow color).

Coliclones is a powder, which includes specific im-munoglobulins (antibodies) that work against group antigens. These antibodies are formed by monoclonal B-lymphocytes in mouse after injecting in its organism antigens in the form of malignant specific cells.

Coliclones have antibodies with only one peculiarity. That means, they react with only one antigen i. e. they do not lead to non-specific polyagglutination of erythrocytes. This property makes them more preferable than standard serums. It is hard to clear standard serums from other antibodies and that is why non-specific reactions with antigen of the examined blood are possible.

There are 2 coliclones in ABO blood system: anti-A and anti-B.

Serums (or coliclones) are mixed on the board with blood

71

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

According to agglutination test the blood group is defined

serum

blood

І

(αβ)

ІІ

(β)

ІІІ

(α)

ІV

(-)

І (0) – – – –

ІІ ( А) + – + –

ІІІ ( В) + + – –

ІV( АВ) + + + –

Rhesus system

In 1940 K. Landsteiner and I. Winner found one more antigen on the erythrocytes of macaque rhesus and they named it rhesus-factor. Later it was discovered that 85% of all whites has it, and their blood is called rhesus-positive. 15% of humans do not have this gene and their blood is rhesus-negative. In American blacks, the percentage of Rh positives is about 95, whereas in African blacks, it is virtually 100 per cent.

There are six common types of Rh antigens, each of which is called an Rh factor. These types are designated C, D, E, c, d, and e.A person who has a C antigen does not have the c antigen, but the person missing the C antigen always has the c antigen. The same is true for the D-d and E-e antigens. Also, because of the manner of inheritance of these factors, each per-son has one of each of the three pairs of antigens. The type D antigen is widely prevalent in the population and considerably

72

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

There are 2 blood groups in rhesus system:Rh+ - blood has antigen D;Rh- - blood does not have antigen D.

The difference between rhesus and ABO system

1 Agglutinins of ABO system appear on the first months of life and stay in blood for the entire life. Antirhesus-antibodies appear only after sensitization (contact of Rh- patient with Rh+-antigens). This can occur after blood transfusion and during pregnancy.

2 Rh-agglutinins – are insufficient antibodies of IgG class, they have small sizes and they can pass through the placental barrier. Agglutinins α and β are sufficient antibodies of IgM class; they have big sizes and cannot pass through placenta.

Rhesus conflict

Rhesus conflict can occur in 2 cases.1 After blood transfusion (after transfusion of Rh+ blood

to Rh- recipient). The first blood transfusion is not dangerous. The maximal titre of antibodies is after 2-4 months. After this period, transfused erythrocytes are derived out of bloodstream. But antirhesus-antibodies are already in the blood of the pa-tient. And after the second transfusion of Rh+ blood agglutina-tion occurs, haemolysis of erythrocytes, which can lead to hemolytic shock and death.

73

Formation of Anti-Rh AgglutininsWhen red blood cells containing Rh factor are injected

into a person whose blood does not contain the Rh factor—that is, into an Rh-negative person—anti-Rh agglutinins develop slowly, reaching maximum concentration of agglutinins about 2 to 4 months later.This immune response occurs to a much greater extent in some people than in others. With multiple ex-posures to the Rh factor, an Rh-negative person eventually be-comes strongly “sensitized” to Rh factor.

Characteristics of Rh Transfusion ReactionsIf an Rh-negative person has never been exposed to Rh-

positive blood, transfusion of Rh-positive blood into that per-son will likely cause no immediate reaction (fig 3.4).

First transfusion Second transfusion

Figure 3.4 - Rhesus conflict of Rh Transfusion Reactions

However, anti-Rh antibodies can develop in sufficient quantities during the next 2 to 4 weeks to cause agglutination of those transfused cells that are still circulating in the blood-.These cells are then hemolyzed by the tissue macrophage sys-tem. Thus, a delayed transfusion reaction occurs, although it is

74

Agglutination of ery-throcytes after the second transfusion

Formation of antirhe-sus-antibodies in re-cipient’s blood

Transfusion of Rh+ erythrocytes to Rh- recipient

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

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

First pregnancy Second pregnancy

Figure 3.5 – Resus conflict by pregnancy

During second Rh-conflict pregnancy, antirhesus-anti-bodies are passing through placenta in the organism of etus and it will cause the destruction of erythrocytes, which will cause

75

Rh+ erythrocytes en-ters woman’s organ-ism during delivery

Formation of antirhe-sus-antibodies in woman’s blood

Antirhesus-antibodies enters fetus’s organism, haemolysis of Rh+ ery-throcytes of fetus

the death of the fetus and missbirth. If during first pregnancy there is fetoplacental insufficiency, small amount of erythro-cytes can get into the woman’s organism and cause the produc-tion of immunoglobulins. As usual, titre of antibodies increases slowly during several months that are why no serious compli-cations occur. In this case hemolytic anemia of newborn can take place (fig. 3.5).

Erythroblastosis Fetalis “Hemolytic Disease of the Newborn”

Erythroblastosis fetalis is a disease of the fetus and new-born child characterized by agglutination and phagocytosis of the fetus’s red blood cells. In most instances of erythroblastosis fetalis, the mother is Rh negative and the father Rh positive. The baby has inherited the Rh-positive antigen from the father, and the mother develops anti-Rh agglutinins from exposure to the fetus’s Rh antigen. In turn, the mother’s agglutinins diffuse through the placenta into the fetus and cause red blood cell ag-glutination.

Incidence of the DiseaseAn Rh-negative mother having her first Rh-positive child

usually does not develop sufficient anti-Rh agglutinins to cause any harm. However, about 3 per cent of second Rh-positive ba-bies exhibit some signs of erythroblastosis fetalis; about 10 per cent of third babies exhibit the disease; and the incidence rises progressively with subsequent pregnancies.

Effect of the Mother’s Antibodies on the FetusAfter anti- Rh antibodies have formed in the mother, they

diffuse slowly through the placental membrane into the fetus’s blood. There they cause agglutination of the fetus’s blood. The agglutinated red blood cells subsequently hemolyze, releasing hemoglobin into the blood. The fetus’s macrophages then con-vert the hemoglobin into bilirubin, which causes the baby’s skin to become yellow (jaundiced).The antibodies can also at-tack and damage other cells of the body.

76

Clinical Picture of ErythroblastosisThe jaundiced, erythroblastotic newborn baby is usually

anemic at birth, and the anti-Rh agglutinins from the mother usually circulate in the infant’s blood for another 1 to 2 months after birth, destroying more and more red blood cells. The hematopoietic tissues of the infant attempt to replace the hemolyzed red blood cells. The liver and spleen become greatly enlarged and produce red blood cells in the same man-ner that they normally do during the middle of gestation. Be-cause of the rapid production of red cells, many early forms of red blood cells, including many nucleated blastic forms, are passed from the baby’s bone marrow into the circulatory sys-tem, and it is because of the presence of these nucleated blastic red blood cells that the disease is called rythroblastosis fetalis. Although the severe anemia of erythroblastosis fetalis is usu-ally the cause of death, many children who barely survive the anemia exhibit permanent mental impairment or damage to motor areas of the brain because of precipitation of bilirubin in the neuronal cells, causing destruction of many, a condition called kernicterus.

Treatment of the Erythroblastotic NeonateOne treatment for erythroblastosis fetalis is to replace the

neonate’s blood with Rh-negative blood. About 400 milliliters of Rh-negative blood is infused over a period of 1.5 or more hours while the neonate’s own Rh-positive blood is being re-moved. This procedure may be repeated several times during the first few weeks of life, mainly to keep the bilirubin level low and thereby prevent kernicterus. By the time these trans-fused Rh-negative cells are replaced with the infant’s own Rh-positive cells, a process that requires 6 or more weeks, the anti- Rh agglutinins that had come from the mother will have been destroyed.

The formation of antibodies in the organism of Rh- - woman can be depressed or totally inhibited with the help of

77

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

Other systems of blood

In 1930 for the discovery of blood groups K. Landsteiner was granted with the Nobel Prize. During ceremony he said that new agglutinogens will be discovered in future and the quantity of blood groups will increase until it reaches the num-ber of Earth’s population. And he was right. Even in ABO blood system several variants of every agglutinogen was found. There are 10 variants of agglutinogen A. The difference between them is that A1 is the most active, and others have less antigenic properties. The blood group of these patients can be mistakenly defined as 1 blood group that can cause complica-tions during transfusion.Except ABO blood system, the most important are Rh, MNS, P, Luteran, Kell-Chelano, Daffi, Diego, and Kid. Antigens of these blood systems are situated on erythrocytes, independently on ABO system and independently on each other. These sys-tems are important only when there are often blood transfu-sions and during pregnancy, which is incompatible by any of these antigens.

78

Blood transfusion

It should be mentioned that blood transfusion is the trans-plantation of non-self tissue. And the first of complications is the immune conflict. The doctor, who made this operation, has the whole responsibility for the consequences, so he should be carefully watching after every stages of it.The stages of blood transfusion:

First stage: The definition of donor’s and recipient’s blood group by ABO and Rh systems.

Second stage: Individual compatibility test. Its objective is to check compatibility for other antigens and antibodies.

Straight test – donor’s erythrocytes + recipient’s blood plasma. This test allows finding antibodies in recipient’s serum for donor’s erythrocytes.

Reverse test – donor’s blood plasma + recipient’s ery-throcytes. This test allows finding antibodies in donor’s blood for recipient’s erythrocytes.

Absence of agglutinations in both tests confirms compati-bility.

Third stage: Biological test.Its objective: proteins compatibility test. 25 ml of donor’s

blood is injected intravenously into the recipient and reaction of the patient is observed.

Complications for the blood transfusion are pains in the lumbal region, darkening of the eyes, increase in body temper-ature.

If blood is transfused into the patient without conscience, then blood pressure, pulse and pupils are observed. Blood pres-sure should not change; pupils should be in normal state. The increase in blood pressure for 20 mmHg is the signal for the transfusion stoppage.

79

Rules of blood transfusion1) The blood should have the same group and same rhesus. Be-

fore, people with I(O) blood group are thought to be univer-sal donors. Their blood was used to transfuse to the recipi-ents of other groups. Now these blood transfusions are not allowed. Why? Erythrocytes of I group do not have antigens A and B and in fact packed red cells of I group can be trans-fused to the recipients of other group. But plasma of I blood group includes agglutinins α and β, that’s why it can cause agglutination in blood, which includes agglutinogens A and B (blood with II, III and IV group). This plasma can be in-jected to the recipients in limited quantities, so transfused agglutinins will be dissolved and agglutination will not oc-cur.

2) Blood should not be injected more than 500 ml of blood at once.

3) Blood should not be transfused from the same donor more than once.

Indication of Blood Transfusion: Decreased blood volume (20% is lost):e.g. in haemor-

rhage. Decreased R. B. Cs.in anemia when Hb % is below 40%. Decreased W. B. Cs. (leucopenia). Decreased blood platelets (thrombocytopenic purpura). Decreased coagulation factors VIII, IX, and XI in

haemophilia. In erythroblastosis fetalis: exchange transfusion to replace

the infant’s blood with Rh-ve group O blood.

Dangers of blood transfusion:I Dangers of Incompatibility:

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

80

a) blocks the capillaries and causes severe pain;b) Blocks the blood vessels, e.g. of the heart causing my-ocardial infarction or thr brain causing paralysis.

2) hemolysis of the donor’s R.B.Cs. leads to jaundice due to excess formation of bilirubin;

3) hypotension: fall of arterial blood pressure due to V.D. caused by histamine released from hemolysed R.B.Cs;

4) decreased urine volume (oliguria) or even anuria and death may occur from renal failure, caused by:

a) hypotension;b) precipitation of Hb (acid hematin) and blocking of re-nal tubules causing anuria;c) fatal hyperkalaemia may result from hemolyssis of R.B.Cs. and failure of K + excretion due to renal failure;d) fatal hypokalaemia, may occur during recovery, since K filtered from the glomeruli is not reabsorbed by dam-aged renal tubules.

II Transmsssion of Diseases:As syplilis, malaria, viral hepatitis or AIDS.

III Allergic reactions:Rigors and fever may occur due to presence of pyrogens.

IV Transmsssion of excessive amount of blood:This may cause an overload and leads to heart failure.

V Tetany:Increased neuromuscular excitability due to decreased calcium

level in plasma. It may occur due to excessive amount of cit-rate in the transfused blood.

81

Questions for self-control1 Definition of agglutinogens, agglutinins, agglutination.2 The characteristics of blood groups by ABO system.3 Modern ideas about blood groups of ABO system.4 The definition of blood groups by ABO system.5 The characteristics of blood groups by rhesus system.6 The definition of rhesus-conflict.7 Other blood systems.8 Stages of blood transfusion.9 Rules of blood transfusion.

Tests for self-control1 Erythrocytes of which blood group don’t have agglutinogens

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

2 Erythrocytes of which blood group have agglutinogens B on their surface:a) group I;b) group ;c) group III;d) group IV;e) there is no such a blood group.

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

82

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

5 Blood plasma of which group doesn’t have agglutinins anti-B:a) group I;b) group II;c) group III;d) group IV;e) there is no such a blood group.

6 During the blood test of patient’s blood by ABO system ag-glutination occurred in standard serums of I and III blood group. Name the patient’s blood group:a) group I;b) group II;c) group III;d) group IV;e) inaccuracy occurred and test should be repeated.

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

83

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

9 Blood group II in ABO system is characterized by the ab-sence of:a) agglutinogens A;b) agglutinogens B;c) agglutinins anti-A;d) agglutinins anti-B;e) no correct answer.

10 In what cases is blood transfusion not allowed:a) donor’s blood group is O(I), recipient’s blood group is

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

B(III);c) donor’s blood group is B(III), recipient’s blood group is

AB(IV);d) donor’s blood group is AB(IV), recipient’s blood group

is O(I);e) donor’s blood group is O(I), recipient’s blood group is

AB(IV);f) is allowed in all cases?

11 In what cases is blood transfusion allowed:a) donor’s blood group is B(III), recipient’s blood group is

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

A(II);

84

c) donor’s blood group is B(III), recipient’s blood group is AB(IV);

d) donor’s blood group is AB(IV), recipient’s blood group is O(I);

e) donor’s blood group is O(I), recipient’s blood group is AB(IV);

f) no correct answer?

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

13 Where are agglutinins anti-A located:a) in cytoplasm of erythrocytes;b) on the surface of the erythrocytes membrane;c) in blood plasma;d) in cytoplasm of leucocytes;e) in the nucleus of leucocytes;f) inside the thrombocytes;g) no correct answer?

14 What do erythrocytes of Rh+ (rhesus positive) blood have on their surface:a) A-antigen;b) B-antigen;c) C-antigen;d) D-antigen;e) E-antigen;f) G-antigen;

85

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

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

86

CHAPTER 4 Protective functions of the blood. Leucocytes

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

Allocation of leucocytes in the organism

There are three locations of the leucocytes in the organ-ism: red bone marrow (30%), peripheral blood (20%) and pe-ripheral tissues (50%).

I Red bone marrow. There are 4 pools of leucocytes:- pool of stem cells, which are in rest state. It is insignifi-cant and it is the reserve for blood formation;- mitotic pool. These cells are in division state;- maturating pool. These cells are differentiating; their maturating period is 3-5 days;- reserve pool. These are mature leucocytes, which can en-ter the bloodstream.

87

II Peripheral blood. There are 2 pools of leucocytes:- pool of circulating leucocytes (50%);- parietal (marginal) pool (50%).

III Peripheral tissues have:- migrating leucocytes;- leucocytes in rest state.

Functional properties of leucocytes

1 White blood cells, which have nucleus and other subcellular structures.2 External membrane is negatively charged.3 They are able to produce amoeboid movement, so they can move not only by the bloodstream, but also against the blood-stream. White blood cells move through tissue spaces by ame-boid motion.

Both neutrophils and macrophages can move through the tissues by ameboid motion. Some cells move at velocities as great as 40 mm/min, a distance as great as their own length each minute.4 They are able to pass through the vessel wall out of the bloodstream into the tissues. This ability is called diapedesis. Leucocytes are circulating in the bloodstream for 4-72 hours, than they stay in tissues. Blood is an intermediate medium for the leucocytes existence.

White Blood Cells enter the tissue spaces by diapedesis. Neutrophils and monocytes can squeeze through the pores of the blood capillaries by diapedesis. That is, even though a pore is much smaller than a cell, a small portion of the cell slides through the pore at a time; the portion sliding through is mo-mentarily constricted to the size of the pore.

5 Chemotaxis. Many different chemical substances in the tis-sues cause both neutrophils and macrophages to move toward

88

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

Figure 4.1 - Movement of neutrophils by diapedesis through capillary poresand by chemotaxis toward an area of tissue damage.

89

Movement of neutrophils by diapedesis through capillary pores and by chemotaxis toward an area of tissue damage6 They have high fermentative activity due to presence of en-zymes-hydrolases, polypeptidases, peroxidases, lipases.7 They synthesize substances, which neutralize toxins.8 They are able to absorb substances on their surface and transport them.9 They have phagocytic activity.10 The term of life – few hours to few days (the shortest term – granulocytes – minutes, hours, maximum 8-10 days; the long-est term – T-lymphocytes – months, years).

Life of white blood cells

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

Neutrophils — 2-5 daysEosinophils — 7-12 daysBasophils — 12-15 daysMonocytes — 2-5 daysLymphocytes — 1/2-1 dayThe amount in peripheral blood – 4-9 · 109/liter.

The decrease of leucocytes amount is called leucopenia, the in-crease – leucocytosis.There are 2 types of leucocytes:

I Physiological – is normal, physiological reaction of the or-ganism in some irritations. There are following types, de-pendently on their causes:1) emotional leucocytosis (occurs in result of emotional

stresses);

90

2) myogenic (occurs in result of intensive physical exer-cises);

3) static (occurs in result of change of the position of the human body from horizontal to vertical);

4) alimental (occurs during or after eating);5) painful (occurs during strong painful feelings);6) leucocytosis of pregnant;7) leucocytosis of newborn.

II Pathological (reactive) – it is connected with the patho-logical process in the organism. Its reasons:

1) infectious diseases;2) inflammatory processes;3) allergic reactions;4) intoxications of endo- and exogenous origin.

The difference between physiological and reactive leucocytosis

Physiological leucocytosis:1) it is redistributing (leucocytes from the parietal pool

are moving into circulation); 2) it has transient character (it is normalizing fast after

the cause disappears);3) leukogram does not change (the correlation between

different forms persists);4) degenerative forms of leucocytes do not appear.

Reactive leucocytosis is connected with the increase of proliferation and maturating of leucocytes in red bone marrow or increase of moving of reserve leucocytes from RBM to the blood. During pathological leucocytosis the correlation be-tween different forms of leucocytes is disturbed.

91

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

Leukogram of a healthy adult human:

LeucocytesGranulocytes Agranulocytes

baso

phils

eosi

noph

ils

neutrophils

lym

phoc

ytes

mon

ocyt

es

immature stab segmented

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

Healthy human has instant leukogram and any changes in it – is the signal to different sicknesses. The disturbance in cor-relation between immature and mature forms of neutrophils is called shift of leukogram. There is shift to the left and shift to the right.

Shift to the left is characterized byincrease in the content of immature neutrophils. Myelocytes appear in blood, the amount of metamyelocytes increase. This happens during leu-cocytosis.

Shift to the right is characterized with domination of ma-ture neutrophils with big amount of segment (5-6) on the back-ground of disappearance of immature forms. This testifies the development of inflammatory process.

The correlation between mature and immature forms of neutrophils is Bobrov’s index:

92

The important index is correlation between neutrophils and lymphocytes.

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

4-5 day 5-6 yearsNeutrophils

Lymphocytes І decussation ІІ decussation

Figure 4.2 - Correlation of neutrophils and lymphocytes throughout life

93

Figure 4.3 – Types of leucosyses

94

Main functions of leucocytes

BasophilesGranules of these cells dye with basic stains into blue

color. Granules contain heparin, histamine, serotonin, peroxi-dase, acid phosphatase, histidinecarboxylase (the enzyme of histamine synthesis). Phagocyte activity is low.

Functions of basophiles1 Participate in allergic reactions. Antigen-antibody complex

affects degranulation of basophiles and histamine release. In low concentrations histamine interacts with H1-receptors and causes basic manifestations of allergic reactions: dilatation of blood vessels, increase of vessel wall permeability, irrita-tion of nervous endings, which cause itching, pains, and in-crease of formation and secretion of mucus in respiratory pathways, contraction of smooth muscles in bronchi. In big concentrations histamine interacts with H2-receptors and causes extinction of these reactions due to inhibition of ba-sophiles degranulation.

2 Participate the development of inflammation, especially dur-ing last (regenerative) phase: heparin predicts blood coagula-tion in inflammation source; histamine dilates capillaries that help resolution and healing.

3 Regulation of vessel wall permeability (increased by his-tamine and serotonin).

4 Participates hemostasis (heparin is an anticoagulant, his-tamine causes vessel spasm after damage).

The increase of basophiles amount (basophilia) is rare. It can cause the development of chronic myeloleucosis, hemo-philia, and polycythemia.

95

EosinophilsGranules of these cells dye with acid stains into pink

color. They have phagocyte activity, but due to low eosinophil concentration in blood their role in this process is insignificant. The concentration of eosinophils in blood is variable during daytime, which is defined by hydrocortisone level (the maxi-mum quantity of eosinophils is at night, the minimum quantity – in the morning). They stay in blood for 3-8 hours, then they migrate into the connective tissue, where they perform their functions. They contain granules of two types. Granules of 1st

type have protein, which has arginine; hydrolytic enzymes; peroxidases; histaminases; esterases. Granules of 2nd type have acid phosphatase and arylsulfatase.

Functions of eosinophils1 Taking part in allergic reactions: histaminase splits his-

tamine, which results in termination of allergic reactions; arylsulfatase degrade anaphilaxin; they can synthesize and release factor that inhibits the release of histamine from ba-sophils.

2 Degradation of toxins with protein nature.3 Taking part in neutralization of toxins which are made by

parasites (helminthes).4 Taking part in fibrinolysis (production of plasminogen).5 Delaying the spread of inflammation, decrease the perfor-

mance of inflammation process (due to histamine neutraliza-tion).

The increase of eosinophils amount (eosinophilia) can be ob-served when allergic reactions, bronchial asthma, helminthosis, chronic myeloleucosis, some infantile infections (scarlet fever), after taking some kind of drugs (antibiotics, sulfanilamides).

96

NeutrophilsGranules of these cells can be dyed with acid or basic

stains into pink-violet color. Only 1% out of all neutrophils is circulating in the bloodstream, others are in tissues. They con-tain 2 types of granules. Primary granules have hydrolytic en-zymes (acid phosphatase, β-glucuronidase, acid protease, aryl-sulfatase); myeloperoxidase, lizocim.

Secondary granules contain basic phosphatase, main cationic proteins, phagocytins, lactoferin, lizocim, aminopepti-dase.In dependence of age they have different shape of nucleus. In dependence of nucleus shape there are immature, stab and seg-mented neutrophils. They circulate in blood for 4-8 hours, and then they move into tissues, where they live 4-5 days.

Functions of neutrophils1 Phagocytosis. Neutrophils are important elements of non-

specific protection of an organism. They are the first to come to infection source or damage spot. Neutrophils neu-tralize not self agents with the help of their enzymes. Neu-trophils can die there. Dead neutrophils form pus.

For the characteristic of phagocytic activity of neu-trophils the following indexes are used: The percent of cells which work as phagocytes (normally

– 68,5 - 99,3%). Phagocytic index (the number of agents, which 1 cell can

consume, normally – 12 - 23);2 Secretion of germicide substances (lysosomal cationic pro-

teins, histones, lactoferin).3 Antiviral activity (production of interferon).4 Stimulation of tissue regeneration after damage (synthesis of

acid glycosaminoglycans).

97

5 Participate in specific immunity by affecting the ac-tivity of T- and B-lymphocytes, increasing the amount of antibodies.

6 Secretion of substances that dilate blood vessels.7 Sexualization of the blood. Most of neutrophils of

women have satellites of nucleus that are surrounding the nucleus. One of X-chromosome is located there. That is why they are called sex chromatin (Bar’s bod-ies). Presence or absence of these satellites allows defining possible sexualization of the blood.

The mechanism of phagocytosisPhagocytosis – is active devourment of the solid sub-

stances by cells. Cells, which are capable of phagocytosis, are called phagocytes. There are poly phagocytes (neutrophils) and mononuclear phagotyces (monocytes).Phagocytes must be selective of the material that is phagocy-tized; otherwise, normal cells and structures of the body might be ingested. Whether phagocytosis will occur, depends espe-cially on three selective procedures. Firstly, most natural struc-tures in the tissues have smooth surfaces, which resist phagocy-tosis. But if the surface is rough, the likelihood of phagocytosis is increased. Secondly, most natural substances of the body have protective protein coats that repel the phagocytes. Con-versely, most dead tissues and foreign particles have no protec-tive coats, which make them subject to phagocytosis. Thirdly, the immune system of the body develops antibodies against in-fectious agents such as bacteria. The antibodies then adhere to the bacterial membranes and thereby make the bacteria espe-cially susceptible to phagocytosis. To do this, the antibody molecule also combines with the C3 product of the complement cascade, which is an additional part of the immune system dis-cussed in the next chapter. The C3 molecules, in turn, attach to receptors on the phagocytic membrane, thus initiating phagocy-

98

tosis. This selection and phagocytosis process is called op-sonization.

Stages of phagocytosis:I Conjugation stage. Phagocyte moves to direction of not self

agent (chemotaxis).II Adhesion stage. Phagocyte interacts with the agent. There

are two mechanisms:1) without receptor: electrostatic and hydrophobic interaction

(phagocyte is negatively charged, positive particles);2) with receptor. On the surface of macrophages there are re-

ceptors for opsonin-substances that can interact with bacte-ria.

III Devourment stage. Its steps: invagination of phagocyte membrane on the contact place; the formation of phagosome, which contains the agent; the formation of phagolysosome: consolidation of phago-

some with lysosomes (secondary granules).IV Digestive stage. Its steps:

The disposal of bacteria – intercellular cytolysis with the help of germicide systems of phagocytes (myeloperoxidase system, which produces hypochloride ion ClO-, free radi-cals and peroxides O30, HO20, OH0, lisocim, lactoferin, non-enzymatic cationic proteins, lactic acid).

Digestion – hydrolysis of killed bacteria with the help of hydrolytic enzymes.

MonocytesMonocytes are the largest blood cells, which does not

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

99

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

Functions of monocytes1 Participation in the development of inflammatory process. Monocytes appear in the inflammation site after neutrophils. They perform phagocytosis in acid medium, where neutrophils loose their activity. In inflammation site monocytes englobe germs, dead leucocytes, damaged cells. They clean the inflam-mation site and prepare it for regeneration. Monocytes are called “organism cleaners” for performing this function.2 Participating in the process of regeneration. Monocytes re-lease factors, which stimulate growth of endothelial and smooth-muscles cells, also they release fibrinogenic factor, which increases the rate of collagen synthesis.3 Formation of “protection wall” around not-self bodies, which can not be destroyed by enzymes.4 Participation in formation of specific immunity. Monocytes englobe, transform and present antigen to immunocompetent cells (T and B-lymphocytes); they participate cooperation of T and B-lymphocytes.5 Antitumoral and antiviral action, which is provided by the secretion of lizocim, interferons, elastase, collagenase.6 Participation in the development of fever. They release en-dogenic leucocytory pyrogen (interleukin-1), which affects the thermoregulation center and causes the increase of body tem-perature.7 Participating in the process of complement formation.8 Participating in blood formation. Monocytes form inter-leukins, which affects leukopoesis.9 Participating in metabolism of lipids and iron.

100

The definition of mononuclear phagocytory system (MPS)

MPS includes: osteoclasts; monocytes; macrophages of connective tissue; macrophages of liver and spleen; macrophages of RBM; macrophages of the lungs; microgliocytes.

The main features of cells of this system are:1) phagocyte activity;2) the presence of receptors for antibodies and comple-

ment;3) common origin and morphology.

Reticuloendothelial system (RES)RES consists of tissue macrophages (fixed monocytes)

which are present in lymph nodes, spleen, Liver, bone marrow and connective tissue.

Functions of RES:1) Defence: The defensive mechanism include:

a) Formation of antibodies against the invading agents (immune response).

b) Phagocytosis and digestion of bacteria and protozoa.c) Engulfing foreign particles, e.g. dust and carbon.

2) Rapair: of tissue after inflammation by removal of dead tis-sue and provide protein and fat needed for repair.3) Blood formation: reticuloendothelial cells (RECs) of bone marrow and spleen may change to haemocytoblasts to form blood cells.4) Removal of old blood cells: from circulation and formation

of bile pigments.

101

5) Storage of iron: needed for erythropoiesis.

Spleen: It is the largest lymphoid organ. Functions:

1) defence: a) phagocytosis: by the RECs in the spleen;b) antibody formation: lymphocytes and plasma cells (immune response).

2) blood cell formation: a) erythropoiesis: In the middle 1/3 of intra-uterine

life. If adult bone is diseased or destroyed, reticuloen-dothelial cells in the spleen is changed to haemocyto -blasts to form blood cells (extramedullary haemopoiesis);

b) lymphopoiesis: Splenic lymph nodes form lym-phocytes and plasma cells. They play an important role in immune response;

c) iron storage needed for erythropoiesis.3) removal of old cells:

a) old RBCs, leucocytes and platelets are removed by RECs of spleen;

b) Hb breakdown and bile pigment formation. Hypersplenism: is a congenital condition in which spleen engulf-the normal blood cells leading to anaemia and thrombocytopenic purpura.4) Blood Reservoir:

In animals (dog & cat), the blood sinuses can acco -modate 1/4 of the blood volume, but much less in man (about 200 ml). The stored blood is squeezed out of spleen to general circulation by contraction of the plain muscle fibres of splenic capsule (supplied by sympathetic nerves) in response to: O2 lack which stimulates the sympathetic nerves.

Haemorrhage.

102

Muscular exercise, hot climate, emotions or adrenaline release.

Lymph nodes:They lie between lymph vessels efferent lymph

vessels finally into thoracic duct which opens into the junction of the left subclavian with internal jugular veins venous circulation.

Macrophages in the Lymph Nodes

Essentially no particulate matter that enters the tis-sues, such as bacteria, can be absorbed directly through the capillary membranes into the blood. Instead, if the particles are not destroyed locally in the tissues, they en -ter the lymph and flow to the lymph nodes located inter-mittently along the course of the lymph flow. The foreign particles are then trapped in these nodes in a meshwork of sinuses lined by tissue macrophages.

Function of lymph nodes1 Filtration: Removal of foreign particles e.g. carbon par-ticles. 2 Removal of bacteria: The lymph nodes enlarge (macrophages increase mucn in number). They are the defence mechanism against local infection.

3 Formation of lymphocytes and plasma cells: which leave the nodes through the efferent lymph vessels to reach the blood stream.

4 Production of antibodies: by lymphocytes as a defence mechanism.

Liver:

Bacteria pass through gastrointestinal mucosa por-tal blood phagocytosis by kupffer cells lining blood si -

103

nuses of the liver and no I bacteria pass to systemic cir -culation.

Macrophages (Kupffer Cells)in the Liver Sinusoids

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

104

Figure 4.5 – Kupffer cells lining the liver sinusoids, showing phagocytosis of India ink particles into the cytoplasm of the Kupffer cells.

LymphocytesUnlike other leukocytes, lymphocytes live not for few

days, but for few years, some of them live throughout human’s life.

Role of lymphocytes in the organism1 They are the central element of immune system; they are re-sponsible for the formation of non-specific immunity.2 They perform the role of “censorship” in the organism: they provide the protection of every non-self agent; provide genetic constancy of the internal medium.3 They provide rejection of transplants reaction.4 They neutralize own mutated cells.

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

105

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

The main function of T-lymphocytesis cellular immune reactions

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

106

The main function of B-lymphocytes is providing hu-moral immune response by synthesis of antibodies. After con-tacting antigen B-lymphocytes migrate into secondary lym-phatic organs, there they multiply and transform into plasmatic cells that are able to produce 5 types of immunoglobulins: IgM, IgG, IgE, IgA, and IgD.

There few forms of B-lymphocytes.1 B1 lymphocytes – are antecessors of antibody-forming cells.2 B2 lymphocytes. These are B-suppressors. They suppress

development and transformation of T- and B-lymphocytes into effector cells.

3 B3 lymphocytes. These are B-killers, which have cytotoxic activity.

O-lymphocytes do not pass through differentiation and in case of necessity can transform into T- and B-lymphocytes. K-cells (killer-cells) and natural killer cells (NKC) also belong to O-lymphocytes; they are responsible for non-specific re-sistance and are especially active against tumor cells.

Regulation of formation and activity of leucocytesRegulation of leucocytes formation is performed by leu-

copoietins and inhibitors of leucopoiesis. Stimulators of leu-copoiesis are leucopoietins. The main source of leucopoietins is activated macrophages. Interleukin-1, factor of tumoral necrosis, colony-stimulating factor belong to leucopoietins. Colony-stimulating factor is the most learned which stimulates the formation of granulocytes in RBM. Interleukin-1 increases transport of reserve leucocytes from RBM into the blood-stream.

Inhibitors of leucopoiesis are high-molecular inhibitors of blood serum – lipoprotein, lactoferin, and keylons. Migra-tion of leucocytes into tissues is regulated by local factors-chemotaxins. Interaction between leucocytes is regulated by cytokins.

107

There are two types of cytokins1 Lymphokins. They are formed in lymphocytes. These are cy-

totoxins, chemotaxins, and mitogens.2 Monokins. They are formed in monocytes, macrophages.

These are interleukin-1, factor of tumoral necrosis, colony-stimulating factor.

108

Questions for self-control

1 Structure of the protective system of the organism.2 Location of leucocytes in the organism.3 Functional properties of leucocytes.4 Definition of leucopenia and leucocytosis. Types of leucocy-

tosis.5 Leukogram. Definition of leukogram shift to the right and to

the left. Definition of leukogram decussation.6 Main functions of leucocytes.7 Mechanism of phagocytosis.8 Definition of mononuclear phagocytic system.9 Regulation of formation and activity of leucocytes.

Tests for self-control

1 Which blood cells are leucocytes:a) monocytes;b) lymphocytes;c) thrombocytes;d) eosinophils;e) basophils;f) erythrocytesg) neutrophils?

2 Which cells provide specific (immune) protection from non-self agents:a) monocytes;b) lymphocytes;c) thrombocytes;d) eosinophils;e) basophils;

109

f) erythocytesg) neutrophils;h) no correct answer?

3 Which blood cells are granulocytes:a) monocytes;b) lymphocytes;c) thrombocytes;d) eosinophils;e) basophils;f) erythrocytes;g) neutrophils;h) no correct answer?

4 Which blood cells are capable for phagocytosis:a) monocytes;b) thrombocytes;c) B-lymphocytes;d) T-lymphocytes;e) erythrocytes;f) neutrophils;g) no correct answer?

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

6 What are the functions of T-lymphocytes:a) transport of oxygen;b) participating blood stoppage;

110

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

7 Which of cells provide immunoglobulin synthesis:a) B-lymphocytes;b) T-lymphocytes;c) O-lymphocytes;d) macrophages;e) neutrophils;f) eosinophils;g) basophils;h) no correct answer?

8 What is leukogram:a) percent of leucocytes among all formed blood elements;b) absolute content of particular forms of leucocytes in unit

of volume;c) percent of mature form of leucocytes among their ante-

cessors;d) percent correlation between particular forms of leuco-

cytes in peripheral blood;e) no correct answer?

9 Which fraction of blood plasma proteins do antibodies be-long to:a) albumins;b) α1-globulins;c) α2-globulins;d) β-globulins;e) γ-globulins;f) no correct answer?

111

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

112

CHAPTER 5 Haemostasis

Haemostasis system is the system, which provides main-tenance of the blood fluidity and stoppage of bleeding after in-jure of blood vessels.

Functions of haemostasis system1 Provides maintenance of the blood fluidity.2 Stops the blood when vessel wall is damaged.

Structure of haemostasis system1 Blood vessels walls.2 Formed blood elements.3 Biochemical systems of blood plasma:

system of blood coagulation; anticoagulative system; fibrinolytic system; calicrein-kinine system.

Mechanisms of haemostasis1 Vessel-thrombocytory (primary, microcirculatory).

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

The role of vessel wall in haemostasisI Vessel wall participate activation of haemostasis with the help of following mechanisms:

113

1) Unmasking of the collagenWhen blood vessel wall is damaged the collagen is un-

masked. So it becomes available for the formed blood ele-ments. Collagen provides contact activation of thrombocytes and Hageman’s factor (F. XII), which initiates the internal mechanism of blood coagulation.

2) Release of ADPADP is released from the damaged cells of vessel wall,

and it is powerful activator of thrombocytes adhesion and ag-gregation.

3) Release of tissue thromboplastinThromboplastin is released from damaged cells of vessel

wall and initiates external mechanism of blood clotting and for-mation of small amount of thromboplastin in the damaged place.

4) Release of Willebrant factorEndotheliocytes of vessel wall form Willebrant factor –

glycoprotein, which participates thrombocytes adhesion.

II Vessel wall provides maintenance of blood fluidity (throm-boresistancy) with the help of following mechanisms:

1) Formation of prostacyclin. Prostacyclin is formed by en-dotheliocytes (derivative of arachidonic acid), which is powerful inhibitor of thrombocytes aggregation.

2) Formation of antithrombin III – powerful natural antico-agulant.

3) Ability of endothelium to fix heparin-antithrombin III complex on its surface, which increases activity of this complex in hundreds times.

4) Formation of fibrinolysis activators.5) Endothelial Surface Factors. Probably the most impor-

tant factors for preventing clotting in the normal vascular system are the smoothness of the endothelial cell surface, which prevents contact activation of the intrinsic clotting

114

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

Role of thrombocytes in haemostasis

Thrombocytes of the peripheral blood are the fragments of cells – megacariocytes, which in bone marrow break down into 3-4 thousands of small parts of blood platelets.

Thrombocyte does not have nucleus and most of subcel-lular structures. At the same time it has complicated structure and well adapted to the functions that it should perform. There are a lot of biologically active substances on the membrane and inside thrombocyte: partially formed by thrombocyte itself and part of them get into it from the blood plasma. Most of sub-stances are in granules. There are 4 types of granules:1st type: include such non-protein structures as ATP, ADP, serotonin, pyrophosphate, adrenalin, calcium.2nd type: include low-molecular proteins, Willebrant factor, fib-rinogen.3rd and 4th type: include enzymes.In their cytoplasm are such active factors as actin and myosin molecules, which are contractile proteins similar to those found in muscle cells, and still another contractile protein, throm-bosthenin, that can cause the platelets to contract; residuals of both the endoplasmic reticulum and the Golgi apparatus that

115

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

Functions of thrombocytes1 Angiotrophic function. Daily 10-15% of all thrombocytes that circulate in blood are used as a nourishment supply of the vessel wall. They adhere to the endothelium, degrade and their content outpour on endothelium. The main component of this

116

content is thrombocytory growth factor, which harden vessel wall, especially the wall of capillaries.

If endothelial cells lose their endothelial nourishment (oc-curs when thrombopenia), they become dystrophic and erythrocytes are able to pass through it. Diapedesis of erythrocytes can be very intensive. Externally it shows up as small hemorrhages – petechiae.

2 Transport. Thrombocytes are able to absorb biologically ac-tive substances and transport them.3 Participating blood clotting. There are a number of sub-stances in thrombocytes, which participate blood clotting. These are thrombocytory (platelet) factors. The most important are:

a) Factor 3 (thrombocytory thromboplastin). It is phospho-lipids, which is released after thrombocytes degradation and it is used as matrix for the reactions for the first phase of clotting.

b) Factor 4 (antiheparin factor) – binds heparin, increases coagulation rate.

c) Factor 5 (fibrinogen) provides compression and contrac-tion of blood clot.

d) Factor 6 (thrombostenin) protein, which is like ac-tomiosin of skeletal muscles has ATPase activity.

e) Factor 10 (pressor factor) – serotonin, which is absorbed by thrombocytes in blood.

f) Factor 11 (aggregation factor) – ADP and thromboxan A2

that provide aggregation.4 Participating stoppage of bleeding. It is determined by the ability of thrombocytes to adhesion and aggregation, which leads to the formation of thrombocytory cork.

117

Vessel-thrombocytory haemostasis

Vessel-thrombocytory haemostasis is performed in several stages:

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

I Spasm of arteries is the contraction of vessels when they are damaged

There are two types of vessel spasm:1 Primary. It has reflex origin. Appears in first seconds of

damage with the help of sympathetic nervous system.2 Secondary. Due to release of biologically active substances

in the damage spot (serotonin, adrenalin, thromboxan). Ap-pears in few seconds after damage.Spasm of vessels develops fast enough and disappears in few seconds and bleeding continues.

The nervous reflexes are initiated by pain nerve impulses or other sensory impulses that originate from the traumatized vessel or nearby tissues. However, even more vasoconstriction probably results from local myogenic contraction of the blood vessels initiated by direct damage to the vascular wall. And, for the smaller vessels, the platelets are responsible for much of the vasoconstriction by releasing a vasoconstrictor substance, thromboxane A2. The more severely a vessel is traumatized, the greater the degree of vascular spasm. The spasm can last for many minutes or even hours, during which time the pro-cesses of platelet plugging and blood coagulation can take place.

II Adhesion of thrombocytes – is the thrombocytes to the damaged vessel wall.

118

The main reason of adhesion is unmasking of colla-gen.

There are 2 stages of adhesion:1) Precontact stage. It is associated with the

changes in shape of thrombocytes. They take spheroid shape with 3 to 10 processes.

2) Contact stage. It is associated with the attach-ment of thrombocytes to the endothelium of blood vessels. Thrombocyte can interact with vessel wall directly and also with the help of special protein – Willebrant factor.

Adhesion occurs easier with the help of two factors:1) Reversion of the membrane charge after damage that pro-

vides electrostatic co-operation of thrombocytes with ves-sel wall.

2) Slow down of the blood movement in microcirculatory ves-sels.

III Aggregation of thrombocytes – is the aggregation of thrombocytes in the damage spot and conglutination of them one to another.

The reasons of aggregation are aggregants. Aggre-gants can have thrombocytory origin (those that are released by thrombocytes) and not-thrombocytory (those that are released by other cells or are formed in plasma). The main aggregants are:

1) ADP;2) Thromboxan A2 and arachidonic acid;3) Biogenic amines (adrenalin, serotonin);4) Factor of thrombocytes aggregation;5) Thrombin;6) Thrombospondin.

Stages of aggregation

119

1 Initial aggregation. It is performed at the same time with ad-hesion. The main reason of it is ADP with not-thrombocy-tory origin, which is released from damaged cells.

2 Reverse aggregation. Aggregation, which can be stopped. The main reason of it is thrombocytory ADP, thromboxan A2, arachidonic acid.

3 Irreversible aggregation. Aggregation with the damage of thrombocytes and it cannot be stopped. The reason of it is thrombin.

Mechanism of aggregationAggregation is performed in two stages:

Stage 1 – stage of thrombocytes activation (fig. 5.1).Aggregants increase thrombocyte membrane perme-

ability to calcium. By the concentration gradient calcium enters thrombocytes. Its concentration increases in these cells. Cal-cium causes following effects in thrombocytes:

1) contraction of myofibrils, which leads to formation of processes;

2) increase of hydrolysis rate of ATP with the formation of ADP, which is aggregant;

3) release (secretion) of granules;4) activation of phospholipase A2, which leads to formation

of arachidonic acid and thromboxan A2.

120

Pathogenic Biogenic Factors Other factors amines damage of vessel aggregants

myofibrils increase of release activation contraction ATP hydrolysis of granules of phospholipase A2Figure 5.1 – Mechanism of aggregations

Stage 2 – stage of thrombocytes conglutination.There are two mechanisms of this stage:

1) formation of bridges between thrombocytes, which are formed with Ca2+ and ADP;

2) formation of bridges between thrombocytes, which are formed with plasma proteins. These proteins are aggrega-tion co-factors of plasma. Fibrinogen, albumins, agrexons A and B.

IV Release reaction – is the process of granules secretion in thrombocytes.There 2 types of release reaction:1) reaction of early release, which is performed during adhe-

sion and initial aggregation. Granules of 1 and 2 type are re-leased during this reaction;

121

[Са2+]

2) reaction of late release, which is performed during irre-versible aggregation. Granules of 3 and 4 type are released during this reaction.

V Thrombus consolidation – packing, retraction of thrombus in consequence of which it loses extra water and be-come hard. Contraction of thrombus is performed with the help of protein thrombostenin.

The scheme of vessel-thrombocytory haemostasisVessel wall damage Spasm of arteries

Adhesion and initial aggregation of thrombocytes

Reaction of early release

Reverse aggregation

Irreversible aggrega-tion

Reaction of late release

Thrombus consoli-dation

Coagulatory haemostasis

Basic Theory More than 50 important substances that cause or affect blood coagulation have been found in the blood and in the tissues—some that promote coagulation, called pro-coagulants, and others that inhibit coagulation, called antico-

122

agulants.Whether blood will coagulate depends on the balance between these two groups of substances. In the blood stream, the anticoagulants normally predominate, so that the blood does not coagulate while it is circulating in the blood vessels. But when a vessel is ruptured, procoagulants from the area of tissue damage become “activated” and override the anticoagu-lants, and then a clot does develop.It is the cascade of biochemical reactions, which result in the formation of fibrin. Plasma factors of coagulation participate coagulative haemostasis:

I – Fibrinogen; II – Prothrombin; III – Tissue thromboplastin. (tissue factor); IV – Ca2+ ions; V – Proaccelerin, Ac-globulin, labile factor; VI – Active form of factor V (accelerin); VII – Proconvertin, stable factor; VIII – Antihaemophilic globulin, antihaemophilic fac-

tor A; IX – Christmas’s factor, antihaemophilic factor B; X – Stuart-Prauer’s factor, prothrombinase; XI – Plasma antecessor of thromboplastin, anti-

heamophilic factor C; XII – Hageman’s factor, contact factor; XIII – Fibrinstabilizing factor, fibrinase.All factors of coagulation can be divided into few groups:

a) substrate for the reaction – F. I (fibrino-gen);

b) Ca2+ ions;c) accelerants of reactions: F. V (proac-

celerin), F. VIII (antihaemophilic globulin);d) proteolytic enzymes: F. II, F. III, F. VII,

F. IX, F. X, F. XI, F. XII.

123

Coagulative reactions are based on the reactions of hy-drolysis, which are performed by proteolytic enzymes. Reac-tions occur in phospholipids of membranes of destroyed ery-throcytes and thrombocytes. Factors of coagulation are fixed on membrane with the help of Ca2+ ions.The main stages of blood coagulation are described by Moravic in 1905.

There are 3 stages of blood coagulation:Stage 1 – formation of active prothrombinase.

In response to rupture of the vessel or damage to the blood it-self, a complex cascade of chemical reactions occurs in the blood involving more than a dozen blood coagulation factors. The net result is formation of a complex of activated sub-stances collectively called prothrombin activator.

Stage 2 – formation of thrombin. The prothrombin activator catalyzes conversion of prothrom-bin into thrombin.

Stage 3 – formation of fibrin. The thrombin acts as an enzyme to convert fibrinogen into fib-rin fibers that enmesh platelets, blood cells, and plasma to form the clot itself.

Formation of prothrombinaseProthrombin and Thrombin Prothrombin is a plasma pro-

tein, an alpha2-globulin, having a molecular weight of 68,700. It is present in normal plasma in a concentration of about 15 mg/dl. It is an unstable protein that can split easily into smaller compounds, one of which is thrombin, which has a molecular weight of 33,700, almost exactly one half that of prothrombin. Prothrombin is formed continually by the liver, and it is contin-ually being used throughout the body for blood clotting. If the liver fails to produce prothrombin, in a day or so prothrombin concentration in the plasma falls too low to provide normal blood coagulation. Vitamin K is required by the liver for nor-

124

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

Active prothrombinase is formed with the help of two mecha-nisms:

1) extrinsic that is initiated by phospholipids, which are released from damaged cells of vessels or from connec-tive tissue;

2) intrinsic that is initiated by coagulative factors of blood.Extrinsic (tissue) mechanism – is fast mechanism, last for

5-20 seconds. Reactions occur on membranes of damaged cells. After damage, cells (mainly lysosomes) excrete active enzymes – tissue thromboplastin (F. IIIa) activates procon-vertin (F. VII). F.VIIa with phospholipids of tissues and cal-cium form complex, which activate prothrombinase (F. X). F. Xa with phospholipids, calcium and F. Va (proaccelerin) form complex, which is tissue prothrombinase.

F. ІІІ

F. VІІ F. VІІа

F. Х F. Ха

Са2+ Са2+

Damage to cells

125

Membranes of damaged cells

Tissue prothrombinase activates small amount of throm-bin, which:

1) is used for the thrombocytes aggregation;2) provides formation of receptors on membranes of aggre-

gated thrombocytes that bind F. Xa, resulting in its un-availability for anticoagulants, mainly for antithrombin III.Intrinsic (blood) mechanism – is durable, last for 5-7

minutes. Reactions occur on the membranes of damaged blood cells (thrombocytes, erythrocytes). Collagen fibers initiate the process, they uncover after damage of vessel. Contact of colla-gen with F. XII (Hageman’s factor, contact), provides activa-tion of F. XII. F. XIIa, F. XIa and phospholipids form complex, which activate F. X. F. Xa together with Ca2+ ions and F. Va also form complex, which is blood prothrombinase.

Contact or fermentative activation

F. ХІІ F .ХІІа

F. ХІ F. ХІа

F. ІХ

Са2+ F. Х F. Ха

Са2+ Са2+ Са2+

Factor III of thrombocytes Са2+

F. ІХа + F. VІІІ

126

In pathological case there is third mechanism of pro-thrombinase formation – macrophagal. Endotoxins of bacteria, immune complexes, complement, products of tissues degenera-tion act on macrophages, provide release of active prothrombi-nase from them (F. Xa). This mechanism has adaptive role, be-cause spread of pathogenic factors in the organism is limited by the blood coagulation.

Internal and external mechanisms are associated with each other with the help of calicrein-kinine system.

F. ІІІ, VІІ

F. XІІ, ХІ, ІХ, VІІІ

Phospholipids of cell mem-branes

Factor 3 of thrombocytes

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

blood prothrombinase.Formation of thrombin

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

Prothrombinase + F. V

prothrombin (F. ІІ) thrombin

Са2+

Са2+ Са2+ Са2+

Phoespholipids of cell membranes,

127

Calicrein-kinine

system

F. 3 of thrombocytesThrombin is a protein enzyme with weak proteolytic ca-

pabilities. It acts on fibrinogen to remove four low-molecular-weight peptides from each molecule of fibrinogen, forming one molecule of fibrin monomer that has the automatic capability to polymerize with other fibrin monomer molecules to form fibrin fibers.

So, the result of the second stage is formation of throm-bin.

Formation of fibrin

Fibrinogen is a high-molecular-weight protein (MW = 340,000) that occurs in the plasma in quantities of 100 to 700 mg/dl. Fibrinogen is formed in the liver, and liver disease can decrease the concentration of circulating fibrinogen, as it does the concentration of prothrombin, pointed out above.

Because of its large molecular size, little fibrinogen nor-mally leaks from the blood vessels into the interstitial fluids, and because fibrinogen is one of the essential factors in the co-agulation process, interstitial fluids ordinarily do not coagulate. Yet, when the permeability of the capillaries becomes patho-logically increased, fibrinogen does then leak into the tissue fluids in sufficient quantities to allow clotting of these fluids in much the same way that plasma and whole blood can clot.

Fibrin is formed from fibrinogen under the influence of thrombin. Fibrinogen – is fibrilar protein, which consists of fib-rin monomer and four fibrinopeptides (2A and 2B). Fib-rinopeptides disturb fibrin monomer to polymerize.

Thrombin rifts fibrinopeptides from fibrinogen and form fibrin monomer. Polymerization of fibrin monomer starts and fibrin S is formed, which is soluble. With the help of F. XIII (fibrin stabilizing factor) and calcium in S molecule covalent connections are formed and fibrin S turns to insoluble fibrin I.

128

Fibrinogen (А2В2)

Thrombin (F.ІІ)

Fibrin monomer+2А+2В

Fibrin polymer soluble (fibrin S)

F. ХІІІ F. ХІІІа

Fibrin insoluble(fibrin I)

So, the result of the third stage is frormation of fibrin. The clot is composed of a meshwork of fibrin fibers running in all directions and entrapping blood cells, platelets, and plas-ma.The fibrin fibers also adhere to damaged surfaces of blood vessels; therefore, the blood clot becomes adherent to any vas-cular opening and thereby prevents further blood loss.

Formed blood elements mesh in fibrin fibers. But this ball of fibers is soft. That’s why the next step of the process is consolidation of thrombus. Packing of thrombus occurs due to contraction of protein thrombocystein (PF-6). After retraction clot is packed in 2 times, serum is removed from it and it be-comes compact and plasma can not pass through it.

Platelets are necessary for clot retraction to occur. There-fore, failure of clot retraction is an indication that the number

129

of platelets in the circulating blood might be low. Electron mi-crographs of platelets in blood clots show that they become at-tached to the fibrin fibers in such a way that they actually bond different fibers together. Furthermore, platelets entrapped in the clot continue to release procoagulant substances, one of the most important of which is fibrin-stabilizing factor, which causes more and more cross-linking bonds between adjacent fibrin fibers. In addition, the platelets themselves contribute di-rectly to clot contraction by activating platelet thrombosthenin, actin, and myosin molecules, which are all contractile proteins in the platelets and cause strong contraction of the platelet spicules attached to the fibrin. This also helps compress the fib-rin meshwork into a smaller mass. The contraction is activated and accelerated by thrombin as well as by calcium ions re-leased from calcium stores in the mitochondria, endoplasmic reticulum, and Golgi apparatus of the platelets. As the clot re-tracts, the edges of the broken blood vessel are pulled together, thus contributing still further to the ultimate state of hemosta-sis.

Retraction last for 2-3 hours. After some time clot starts to spring with fibroblasts. This occurs under the influence of thrombocytes growth factor. Integrity of the damage spot of vessel is restored.

130

Figure 5.2 – Red thrombus: erythrocytes and fibrin fiberRole of Calcium Ions in the Intrinsic

and Extrinsic Pathways

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

Fibrinolysis

Fibrinolysis – disintegration of fibrin. Disintegration of fibrin starts at the same time with thrombus retraction. Disinte-gration is performed by fibrinolysis system.

131

Content of fibrinolytic system1 Plasminogen (profibrinolysin) – not-active proteolytic en-

zyme, which is always in blood plasma.2 Plasmin (fibrinolysin) – active enzyme, which is produced

in the result of effect of active proteases on plasminogen.3 Activators of fibrinolysis – substances that are proteases or

provide proteases synthesis.4 Inhibitors of fibrinolysis.

There are internal and external mechanisms of fibrinoly-sis activation.

Internal mechanism includes activation of F. XII and for-mation of calicrein, which cause large amount of fibrinolysis activators to appear in blood.

Internal mechanism is associated with transport of ready fibrinolysis activators into the blood.

Activation of Plasminogen to Form Plasmin: Then Lysis of Clots

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

A Plasmin Inhibitor, Alpha2-Antiplasmin Plasmin not only destroys fibrin threads but also functions as a proteolytic en-zyme to digest fibrinogen and a number of other clotting fac-

132

tors as well. Small amounts of plasmin are formed in the blood all the time, which could seriously impede the activation of the clotting system were it not for the fact that the blood also con-tains another factor, alph2-antiplasmin, t hut binds with plas-min and inhibits it. Therefore, the rule of plasmin formation must rise above a certain critical level before it becomes effec-tive.

Internal Externalmechanism mechanism

Plasminogenfactor XIIa,calicrein

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

Inhibitors

After activation plasmin is blocked by antiplasmin, that’s why it works only locally in the blood clot.

133

Anticoagulative systemBlood fluidity is maintained by several mechanisms such as:1) smooth surface of vessel wall endothelium;2) negative charge of vessel wall and formed

blood elements, so they repel from each other;3) thin fibrin layer on vessel wall, which absorb

factors of blood clotting, especially thrombin;4) synthesis of prostacyclin by endothelium,

which is inhibitor of aggregation;5) ability of endothelium to synthesize and fix

antithrombin III;6) presence of anticoagulants in the bloodstream.

Classification of anticoagulants1 Primary (always present in plasma): antithrom-

bin III, heparin, α1-antithropsin, α2-macroglobulin.2 Secondary (they are formed in the process of

clotting): antithrombin I, products of fibrinolysis.Antithrombin III is α2-globulin of blood plasma. Its

concentration in blood plasma is 240 mg/ml. It is 75% out of all anticoagulative reserves of blood. It inactivates thrombin (F. IIa), XIIa, XIa, Xa, IXa.

Heparin – sulphuretted polysugar. Active only with an-tithrombin III. It provides fixation of antithrombin III on the surface of endothelium that increases its activity in hundred times.

Significance of the Plasmin System The lysis of blood clots allows slow clearing (over a period of several days) of ex-traneous clotted blood in the tissues and sometimes allows re-opening of clotted vessels. An especially important function of the plasmin system is to remove very minute clots from the millions of tiny peripheral vessels that eventually would all be-come occluded were there no way to cleanse them.

134

Questions for self-control1 Functions of haemostasis system.2 Mechanisms of haemostasis.3 Role of vessel wall in haemostasis.4 Functions of thrombocytes.5 Stages of vessel-thrombocytory haemostasis.6 Spasm of vessels. Its types.7 Mechanisms and stages of adhesion.8 Definition of thrombus aggregation. Stages and mechanisms

of aggregation.9 Consolidation of thrombus.10 Factors of blood clotting.11 Stages of blood clotting.12 Formation of tissue and blood prothrombinase. Role of pro-

thrombinase.13 Role of calicrein-kinine system in haemostasis.14 Formation of thrombin.15 Formation of fibrin.16 Fibrinolysis.17 Anticoagulative system.

135

Tests for self-control1 Which substances out of the following are factors of blood

clotting:a) antithrombin III;b) fibrinstabilizing factor;c) bradykinine;d) serotonin;e) angiotensin III;f) calidine;g) magnesium ions;h) no correct answer?

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

3 Which substances are factors of blood clotting:a) heparin;b) histamine;c) profibrinolysin;d) vitamin K;e) vitamin E;f) fibrinogen;g) magnesium ions;h) no correct answer?

136

4 Which substances comprise anticoagulative system:a) antithrombin III;b) proaccelerin;c) fibrinstabilizing factor;d) histamine;e) antihaemophilic globulin;f) bradykinine;g) calcium ions;h) no correct answer?

5 Which substances are factors of blood clotting:a) proconvertin;b) prothrombin;c) Hageman’s factor;d) serotonin;e) calidine;f)antithrombin III;g) calcium ions;h) no correct answer?

6 Which substances comprise fibrinolytic system:a) heparin;b) fibrinogen;c) histamine;d) plasmin;e) proaccelerin;f) vitamin E;g) calcium ions;h) no correct answer?

7 First stage of blood clotting results in the formation of:a) thrombin;b) fibrin;c) calicrein;

137

d) plasmin;e) ptothrombinase;f) no correct answer.

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

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

10 Formation of thrombin is the result of:a) first stage of blood clotting;b) second stage of blood clotting;c) third stage of blood clotting;d) activation of anticoagulative system;e) activation of fibrinolytic system;f) no correct answer.

11 Secretion of heparin is the result of:a) first stage of blood clotting;b) second stage of blood clotting;c) third stage of blood clotting;

138

d) activation of anticoagulative system;e) activation of fibrinolytic system;f) no correct answer.

12 Which enzyme induces formation of thrombin:a) Hageman’s factor;b) tissue thromboplastin;c) fibrinolysin;d) proconvertin;e) calicrein;f) plasmin;g) prothrombinase;h) no correct answer?

13 Which processes participate stoppage of bleeding in vessels with diameter less than 100 mkm:a) spasm of arteries;b) adhesion of thrombocytes;c) aggregation of thrombocytes;d) formation of fibrin;e) no correct answer?

14 Thrombin induces transformation of:a) proconvertin into convertin;b) proaccelerin into accelerin;c) factor V into factor VII;d) factor VII into factor VIII;e) factor VIII into factor IX;f) factor IX into factor X;g) fibrinogen into fibrin.

15 Which are the two mechanisms of bleeding stoppage (haemostasis):a) vessel-thrombocytory haemostasis;

139

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

140

PRACTICAL WORKS

Practical work #1. Definition of hematocrit index

Hematocrit index (hematocrit) characterizes the percent-age volume of formed elements (erythrocytes) in blood. There are venous, capillary and arterial hematocrits, because the vol-ume of formed blood elements, especially erythrocytes, is dif-ferent in different parts of the bloodstream. The lowest hemat-ocrit is in the arterial blood.

Objectives: to define the percentage volume of formed elements (erythrocytes) in capillary blood and estimate its value.Requirements for work: hematocrit capillary or micropipette, centrifuge with 8000 rotations per minute, plasticine, aqueous solution of heparin (with activity 5000 OD/ml), 96% alcohol, 2% alcohol solution of iodine, cotton wool.

to wash calibrated hematocrit pipette with heparin solu-tion;

to blow pipette; to dry pipette and fill with blood on 7/8 long; to fix the opening of pipette with plasticine and to cen-

trifuge for 5 minutes with 8000 rotations per minute; to define the percentage volume of formed elements

comparatively to the general blood volume, that is hematocrit value.

Recommendations for writing down the resultsWrite down hematocrit value in percents.Answer following questions in conclusion:

Is the percentage volume of formed element (erythro-cytes) in capillary blood normal?

What changes in blood system testify increase or de-crease of hematocrit value?

141

Practical work #2. Definition of osmotic resistance of ery-throcytes

Resistance of erythrocytes is the ability of erythrocyte membrane to resist the destructive action of low osmotic pres-sure in solution, where erythrocytes are. In result of such action haemolysis can occur (degradation of the erythrocytes mem-brane).

Resistance of erythrocyte membrane should be studied relatively to hypotonic solutions of sodium chloride. When concentration is 0,54% - 0,45% normally only erythrocytes with the lowest resistance pass through haemolysis (minimal erythrocytes resistance). In case of further decrease of concen-tration of sodium chloride more persistent erythrocytes pass through haemolysis. When concentration is 0,40% - 0,34% of sodium chloride even most persistent erythrocytes break down (maximal resistance). Solution becomes transparent, like var-nish (as called laky solution). Breach between upper and lower limit of resistance is called amplitude of resistance.

Objectives: to define the borders of changes in osmotic pressure of blood plasma, when the integrity of erythrocyte membrane is kept and estimates the durability of the erythro-cyte membrane.

Requirements for work: 7 Vidal’s test-tubes (or plain lab-oratory tubes), holder, 0,5% solution of sodium chloride, distil-lated water, blood, conserved with 5% solution of sodium cit-rate, 3 pipettes with the equal size of the drop openings.

Make following dilutions with 0,5% solution of sodium chloride:

142

№ of test-tybe

Number of 0,5% NaCl drops

Number of dis-tillated water drops

Concentration of solution

1 25 - 0.5%2 24 1 0.48%3 22 3 0.44%4 20 5 0.40%5 18 7 0.36%6 16 9 0.32%7 14 11 0.28%

Add 1 drop of conserved blood in every test-tube. Accurately mix the content of every tube until it will

get steady color and leave it for 1 hour in a holder. In 1 hour time observe the destruction of erythrocyte

membrane (haemolysis). State of blood is estimated by the grade of color of sodium chloride solution, which is up to sedimentated erythrocytes in test-tubes.

Recommendations for writing down the results: Write down the value of minimal and maximal erythro-

cyte resistance. Calculate its amplitude.Answer the following questions in conclusion: Do the borders of changes in osmotic pressure, when

erythrocyte membrane keeps its integrity correspond to normal?

Is the integrity of erythrocyte membrane normal? What testifies increase (or decrease) of the resistance

borders?

Practical work #3. Definition of ESR

143

Blood is a colloid solution and suspension at the same time. Particles of substances that are suspended in liquid medium undergo action of inverse forces: gravity force, which provides sedimentation of particles and diffusion, with the help of which particles of colloids move.

Rate of particle sedimentation is straightly proportional to the square radius and difference between the densities of suspended substance and dissolvent. The charges of particles, which are in the solution, also play a great role.

Formed elements, which are suspended in the solution of colloids and bound with them by charges, will sedimentate in stabilized blood because of their agglomeration. Blood will divide into two layers: upper is plasma, lower is blood ele-ments.

Correlation of cholesterol and lecithin in plasma, con-tent of bilious pigments and bilious acids, changes in sticki-ness, pH, characteristics of erythrocytes, and amount of hemo-globin and so on affect ESR.

Main factors that affect ESR are quantitative and qualita-tive changes of proteins in plasma. So, increase of globulin amount in plasma leads to increase of ESR and decrease of its amount and increase of albumins amount leads to decrease of ESR.

ESR gives information about the correlation of plasma proteins and their electrostatic interaction with erythrocytes.

Normally ESR of men is 2-10 mm/hour, of women – 2-15 mm/hour.

Objectives: to define erythrocyte sedimentation rate; es-timate correlation between albumins and globulins in blood plasma.Requirements for work: Panchenkov’s apparatus, which in-cludes holder with rubber base, capillaries for the definition of

144

ESR, glass, 5% solution of sodium citrate, 96% ethanol, 2% al-cohol solution of iodine, cotton wool.

Figure 6.1 – Panchenkov’s apparatus

Wash the capillary with 5% solution of sodium citrate; Fill the capillary with mark “R” (reagent) with 5% solu-

tion of sodium citrate and put on the glass; Fill the capillary with mark “B” (blood) with blood 2

times and put it on the glass; Mix the blood with solution (correlation 4:1), by filling

the capillary with it and putting it back several times; Fill the capillary with this mixture till the mark “B”; Clean the opening of the capillary from the blood with

cotton wool; Put the capillary into the Panchenkov’s apparatus; Measure the lighten layer of plasma after 1 hour, which

will be the measure of ESR.Recommendations for writing down the results: Draw down the Panchenkov’s apparatus and capillary

for the measurement of ESR. Write down the size of the layer of plasma up to the

erythrocytes, which are sedimentated (mm/hour).Answer following questions in conclusion:

145

Is the ESR normal in the examined blood? Is the correlation between albumins and globulins nor-

mal in the blood plasma? What changes in blood plasma testify the increase of

ESR?Practical work #4. Calculation of the erythrocytes

Erythrocytes are counted with the help of Goryaev’s calculating camera under the microscope. This method is com-plicated but regular enough (permissible variation is not up to 2,5%).

The net rate of calculating camera consists of 225 large quadrates, 25 out of them are divided into 16 small ones.The side of small quadrate is 1/20 mm, square is 1/400 mm2, the height of camera (the distance between the bottom and the covering glass) is 1/10 mm. So, the size of the camera upon the small quadrate is 1/4000 mm3 (1/400•1/10).

Blood for the count of the erythrocytes is diluted in spe-cial mixing tube (melanger) – capillary pipettes with the am-pule dilation. There are marks 0,5 and 101 on the mixing tubes. Mark 0,5 shows what part of the mixing tube takes this column of capillary, filled with blood. This volume takes 1/200 of the all volume of the mixing tube. So, blood is dissolved in 200 times. Blood can be diluted in 200 times by other methods. For example, put 4 ml of 5% solution of sodium citrate in the test-tube and add 20 ml of blood with micropipette. It is necessarily to washout the micropipette three times in this solution, so all blood will get into the tube.

Normally the amount of erythrocytes in men is 4-5•1012, in women 3,9-4,7•1012.

146

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

147

Objectives: to count ery-throcytes; to estimate the quantity of erythrocytes in the peripheral blood.

Requirements for work: microscope, Goryaev’s camera, covering glass, mixing tube for the erythrocytes, 3% solution of sodium chloride, 96% alcohol, 2% alcohol solution of iodine, cotton wool.

Figure 6.3 – Erythrocyte mixing tube

To prepare Goryaev’s camera for work: perform defatting with alcohol and dry the camera and

the covering glass; lap the covering glass to the camera till the appearance

of the Newton’s rings; find the net under the large enlargement.

1. Prepare blood for work: fill the capillary till the mark of 0,5; clean the opening

from blood with the cotton wool; don’t release the blood from the capillary and fill it with

3% solution of sodium chloride till the mark of 101; mix the solution with blood in the ampule of mixing

tube (there is a tiny red ball to ease the mixing process). Blood will be diluted in 200 times.

2. To fill camera with blood: blow first two drops of solution out of the capillary on

the cotton wool;

148

next drops from the ampule solution put in the camera. Put the tip of the melager on the edge of camera near the covering glass and blow it out accurately. Solution will go under the covering glass into the camera and will fill it. Wait for 1-2 minutes for erythrocytes to sedi-mentate on the bottom of the camera.

3. Calculate the amount of the erythrocytes: count the amount of erythrocytes in 5 large quadrates of

the net diagonally. Remember Burker’s rule when counting erythrocytes: in small quadrates count cells, which are inside the quadrate and on its superior and left sides. This will predict counting erythrocytes twice.

calculate the amount of erythrocytes in 1 mkl of blood by formula:

Where E – is the quantity of erythrocytes in 1 mkl;a – the amount of erythrocytes in 5 large quadrates of net;5 – the amount of large quadrates;16 – the amount of small quadrates;200 – the degree of blood dilution;1/4000 mm3 – the volume of 1 small quadrateThere is a simplified formula:E = a•104

- to find the amount of erythrocytes in 1 l of blood by formula E•106

Recommendations for writing down the results:Draw down the mixing tube for the erythrocytes.Write down the process of the erythrocyte counting.Define the amount of erythrocytes in 1 l of blood.Answer following questions in conclusion:Is the amount of erythrocytes normal in the examined blood?

149

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

Sali method is used for the definition of the hemoglobin amount in blood.

Principle of this method is the following. After adding hydrochloric acid hemoglobin transforms into hydrochloric hematin that has brown color, intensity of which is straightly proportional to the hemoglobin content. The solution of hy-drochloric hematin is diluted with water till it gets standard color.

This method of the definition of hemoglobin amount gives results with accuracy to 10%. Mistake is associated with the technical problems during definition and possible subjec-tive mistakes in the definition of color. But this method is widely spread because it is easy and simple.

Hemometer Sali is used for this definition. It is a holder with 3 test-tubes. There is 1% solution of hydrochloric hematin. Middle tube has marks from 0 to 23•10 g/l and it is for the definition of hemoglobin in the examined blood.Normally, the amount of hemoglobin in men is 140-160 g/l, in

women – 120-140 g/l.

150

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

With the help of pipette put 0,1N solution of hydrochlo-ric acid into the middle tube of hemometer till the low-est mark (0,2 ml);

For the definition of hemoglobin put blood into the cap-illary till the mark (0,02 ml);

Clean the opening of the capillary with cotton wool; Put the capillary into the tube with acid and carefully

eject the blood from the capillary to the bottom of the tube;

Without taking out the capillary from the tube, clean it with acid from the upper layers;

Mix blood with acid by shaking the tube; Put the tube into the hemometer; Leave hemometer for 4-5 minutes. By this time acid

will ruin erythrocyte membrane, transform hemoglobin into hydrochloric hematin that has brown color;

Put distillated water into the middle tube, until the color of the solution in the middle tube will become the same color with the one in standard tubes;

Fix the level of solution in the middle tube by the lower meniscus;

To get the value in g/l, the value in g% should be multi-plied by 10.

Recommendations for writing down the results:Draw down hemometer of Sali.Calculate the amount of hemoglobin in the examined blood. Write down the results in the absolute units.

151

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

Practical work #6. Calculation of the color index

In clinics 5 indexes of the blood is calculated: color in-dex, average hemoglobin content in the erythrocyte, average concentration of hemoglobin in the erythrocyte, average ery-throcyte volume, average diameter of erythrocyte. It is neces-sary to define color index in clinical analysis.

The value of this index shows relative hemoglobin con-tent in every erythrocyte. Normally color index is 0,85 – 1,15. Increase or decrease of it testifies interruption of the erythro-cytes saturation with hemoglobin.

a b c

152

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

To calculate hemoglobin amount in % the following op-erations are performed. For example there is 14 g% of hemo-globin in blood:

16,7 g% - 100%14 g% - x x = 14•100 / 16,7 = 84%

Objectives: to define and estimate relative hemoglobin content in the erythrocyte.Find the amount of erythrocytes in 1 mkl of blood and amount of hemoglobin, calculate color index (CI).If the amount of hemoglobin is in g/l, then CI is calculated us-ing a formula:CI = (number of Hb (g/l)•3): (first 3 digits of the erythrocytes amount)For example, if hemoglobin amount is 140 g/l, erythrocytes – 4,2 • 1012 (4 200 000 000 000), then CI = (140 • 3) : 420 = 1Recommendations for writing down the results:Calculate the color index using data from previous practical works.Answer following questions in conclusion:What is the degree of erythrocyte saturation with hemoglobin and what does it testify?

Practical work #7. Calculation of leukocytes

Leukocytes or white bodies have many variations and perform different protective high-specified functions. That’s why the amount of leukocytes varies during a short period of time in the healthy man (from 4•109/l to 9•109/l).

153

Dilution of the blood for the calculation of leukocytes is better to be performed with the help of melanger.

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

Figure 6.6- Melanger for leukocytes

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

1. To prepare Goryaev’s camera for work:

154

perform defatting with alcohol and dry the camera and the covering glass;

lap the covering glass to the camera; find the net under the large enlargement.2. Prepare blood for work: fill the capillary till the mark of 0,5; clean the opening

from blood with the cotton wool; don’t release the blood from the capillary and fill it with

3% solution of acetic acid till the mark of 11; mix the solution with blood in the ampule of mixing

tube (there is a tiny red ball to ease the mixing process). Blood will be diluted in 20 times.

3. To fill camera with blood: blow first two drops of solution out of the capillary on

the cotton wool; the next drop from the ampule solution is put in the

camera. Put the tip of the melager on the edge of cam-era near the covering glass and blow it out accurately. Solution will go under the covering glass into the cam-era and will fill it. Wait for 1-2 minutes for leukocytes to sedimentate on the bottom of the camera.

4. Count the amount of the leukocytes: count the amount of erythrocytes in 100 large

quadrates. For better accuracy calculation should be performed on the whole area of net, starting from the left edge.

calculate the amount of erythrocytes in 1 mkl of blood by formula:

L = (a • 4000 • 20) / 1 • 100 • 16

Where L – the amount of leukocytes in 1 mkl;a – the amount of leukocytes in 100 large quadrates of net;100 – the amount of large quadrates;

155

16 – the amount of small quadrates;20 – the degree of blood dilution;1/4000 mm3 – the volume of 1 small quadrateThere is a simplified formula:L = (a:2) • 102

- to find the number of leukocytes in 1 l of bloodL • 106

Recommendations for writing down the results:Draw down the melanger for leukocytes.Count the amount of leukocytes in 100 quadrates of net.Calculate the number of leukocytes in 1 ml and in 1 l of blood.Answer following questions in conclusion:Is the amount of leukocytes normal in examined blood and what does it testify?

Practical work #8. Definition of the blood group by ABO system with the help of coliclones

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

divide dry white plate on 4 sectors with glass-pencil. make notes “anti-A”, ”anti-B”. with the help of pipettes put drops (0,1 ml) of coli-

clones anti-A and anti-B in the accordant sector. Make

156

a string using the same pipette (diameter of the string should not be less than 1,5 – 2 cm).

put one drop of the examined blood on the glass. using the angles of another glass put blood (0,01 ml) in

the drops of coliclones. Angles of glass should be dif-ferent for different coliclones.

mix blood with the drop of coliclone with the angle of glass. Correlation between blood and coliclone should be 1:10 (mixed drop has pink color).

observe the reaction on the plate during 2,5 minutes.Results and control

1. Agglutination does not occur with coliclone anti-A and with coliclone anti-B. So, examined erythrocytes do not have antigens A and B, and blood belongs to group I (0, αβ).

2. Agglutination occurred only with coliclone anti-A. So, examined erythrocytes have only antigen A and blood belongs to group II (A, β).

3. Agglutination occurred only with colicone anti-B. So, examined erythrocytes have only antigen B and blood belongs to group III (B, α).

4. Agglutination of erythrocytes is observed in both drops of coliclones. So, examined erythrocytes have both antigens A and B and blood belongs to group IV (AB).

It should be mentioned that all processes that occur after 2,5 minutes after mixing will not be connected with specific agglu-tination, which is examined and those can have other reasons. False agglutination can occur when erythrocytes will gather in monetary column. This agglutination can be easily discerned from the real one if added 1-2 drops of isotonic solution of sodium chloride to 1 drop of blood. False agglutination will disappear in this case.Recommendation for writing down the results:

157

Write down the results of agglutination reaction with coliclones anti-A and anti-B in the following chart:

coliclone

bloodAnti-A Anti-B

І (0, αβ)

ІІ (А, β)

ІІІ (В, α)

ІV (АВ)

Answer the following questions in conclusion:What means presence/absence of agglutination of the examined blood with coliclone anti-A?What means presence/absence of agglutination of the examined blood with coliclone anti-B?

Practical work #9. Definition of blood group by ABO sys-tem with the help of standard serums

Objectives: to define a blood group by ABO system.Requirements for work: chart boards for the definition of blood group, pipettes, glass, examined blood, standard serums, iso-tonic solution of sodium chloride.

Put one drop (0,1 ml) of standard serum of I, II, III group on the surface of the chart board in the corre-sponding sector

Put one drop of the examined blood on the glass with the help of a pipette

158

Using another glass put part of blood (0,01 ml) in each drop of blood serum. Angles of glass for every serum should be different.

Mix blood with serum using the same angle of glass. Correlation of blood and serum should be 1:10 (mixed drop should have pink color).

Observe the reaction on the chart board during 5 min-utes.

Results and control1. Agglutination is absent in all drops of serums. So, we

can suggest that examined erythrocytes do not have antigens A and B and blood belongs to group I (0, αβ).

2. Agglutination occurred with serums of I and III group. So, we can suggest that erythrocytes have antigen A and blood belongs to group II (A, β).

3. Agglutination occurred with serums of I and II group. So, we can suggest that erythrocytes have antigen B and blood belongs to group II (B, α).

4. Agglutination is observed in all serums. So, we can suggest that erythrocytes have both antigens A and B and blood belongs to group IV (AB). In this case serum of group IV is put on the plate and if agglutination does not occur, blood belongs to group IV.

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

serum

bloodІ (αβ) ІІ (β) ІІІ (α) ІV(-)

І (0) – – – –

ІІ (А) + – + –

ІІІ (В) + + – –

159

ІV (АВ) + + + –

Answer following questions in conclusion:What means presence/absence of agglutination of the examined blood with serums of different groups?

Practical work #10. Definition of the blood group by CDE system by method of Zotikov-Donsky

Objectives: to define blood group by CDE blood system.Requirements for work: white plate, 4 pipettes, glass, ex-

amined blood, standard rhesus-positive – (Rh+), standard rhe-sus-negative – (Rh-) erythrocytes, standard antirhesus serum, and pencil for glass.

divide the plate with the pencil for glass into two parts. put one drop of standard antirhesus serum on the one

part of plate, make it plane (with diameter less than 2 cm).

put the examined blood on the glass. Using another glass put part of blood in drop of serum and mixes it. Correlation of blood and serum should be 1:10.

There are two variants of reaction:1. If agglutination occurred that means that there are anti-

gens CDE in the examined blood, so blood is rhesus-positive. To confirm the result, control reaction should be done.Put 2 drops of standard serums on the other part of the plate and with the help of glass mix standard erythro-cytes Rh+ and Rh-. If agglutination will not occur in the drop with Rh- erythrocytes and will occur in the drop with Rh+ erythrocytes, that means that serum is reacting

160

correctly. So, results show that blood includes antigen CDE.

2. If agglutination does not occur, than after control reac-tion we can say that blood does not include antigens CDE. So it is rhesus-negative.

Recommendations for writing down the results:Draw and describe observed reaction.Answer following questions in conclusion:What does agglutination mean when examined and controlled; is the examined blood rhesus-positive or rhesus-negative?

Practical work #11. Blood test for blood group compatibil-ity

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

Objectives: to make a test and to estimate blood compati-bility.

Requirements for work: Petri dish, mirror, pipettes, glass, serum (plasma) of recipient, blood (erythrocytes) of donor.

put 1 drop of recipient’s serum (plasma) in Petri dish. Diameter of drop should be not smaller than 1,5-2 cm.

put 1 drop of donor’s blood on the glass and put part of blood into serum using other glass (correlation 1:20).

put Petri dish on the mirror and observe for 5 minutes.Recommendations for writing down the results:

Describe the results of the test.Answer following questions in conclusion:

161

What does agglutination mean in the test; is the donor’s and re-cipient’s blood compatible by not main blood systems?Practical work #12. Bleeding prolongation test

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

rub a finger with 96% ethanol and check the time of the injury. Prick should be done with the whole length of the needle.

clean the blood every 30 seconds with the blotting pa-per.

check the time when no blood will be on the paper.Recommendations for writing down the results:Write down the time when the blood is stopped. Describe changes of the blood splash diameter on the paper after every clean.Answer following questions in conclusion:Is the bleeding duration normal? What does it testify?

Practical work #13. Definition of capillary resistance

Vessel-thrombocytory haemostasis is the first stage of blood clotting. With the help of this process small vessel injuries can be liquidated in short period of time. Thrombocytes constantly control capillaries permeability. Method that is described al-lows defining if thrombocytes perform their protective function when blood pressure is increased. The degree of capillary pro-

162

tection depends on the characteristics of capillary endothelium and the number of thrombocytes in blood.Objectives: to estimate capillaries resistance to the increase of blood pressure.Requirements for work: Rive-Rochi apparatus, clock.

draw a circle with diameter 5 cm on the skin of the in-ternal surface of the forearm.

put the cuff from the manometer on the arm and make the pressure in it 12 – 13,3 kPa (90 – 100 mmHg).

hold the pressure in the cuff for 5 minutes. wait till the blood circulation in the hand would be re-

stored. Count the number of petechiae (small hemor-rhages) on the marked surfaces. Pay attention to the di-ameters of the petechiae.

normally the number of petechiae should not be more than 10 and diameter of it – 1 mm. In case of weak pos-itive reaction – 20-30, strong positive – more than 30 petechiae

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

Practical work #14. Calculation of thrombocytes quantity in blood

There are direct and indirect methods of counting thrombo-cytes. To conduct the last one, they use definition of quantity of blood platelets in the tinted smear. This index correlates with the quantity of erythrocytes and in blood analysis is writ-ten as 1:1000. After this, absolute quantity of thrombocytes is calculated in 1 l of blood. Direct method is more accurate. Ex-amination is performed in Goryaev’s camera immediately after

163

taking the blood. To stabilize the blood, the following solutions can be used: 5-7% trilone B, 1% ammonium oxalate with methylene-blue and others. Calculation methods are also differ-ent: using phase-contrast and luminescent microscopy.Objectives: to calculate and estimate the quantity of thrombo-cytes in the examined blood.Requirements for work: microscope, lighter, mixer for the red blood, Goryaev’s camera, covering glass, holder with test-tube, Petri dish with wet filtrating paper (wet camera). 1% solution of ammonium oxalate, needle, 96% ethanol, 2% alcohol solu-tion of iodine, cotton wool.

Principle for calculation of thrombocytes is similar to the one which is used for the definition of erythrocytes amount. But thrombocytes are degrading fast if we were to retire them from the bloodstream, that’s why the blood should be taken as fast as possible and the mixer should be wet. So, mixer should be par-tially filled with stabilizing liquid from the test-tube. Prick the finger with the needle and put the ending of the mixer into the blood, fill it till the mark 0,5. Check for abcence of air in the end of the mixer, so stabilizing liquid will not drop on the blood on finger. So, mixer should be held horizontally. Fill the mixer with blood, put the ending of the mixer into the test-tube with liquid and fill it till the mark 101. Mix the content of the melanger and leave it in the horizontal position for 10 minutes for haemolysis of erythrocytes to occur. Prepare the calculating camera. Fill it with solution from melanger and put in Petri dish for 5 minutes. It is necessary for the thrombocytes to settle on the bottom. Than take it out of the wet camera, pur under the microscope and calculate thrombocytes in 25 large quadrates of the net (25 • 16 = 400 small quadrates).Recommendation for writing down the results:Number of thrombocytes in 1 mkl is calculated by the follow-ing formula:

164

X = (a • 400 • 200) / 400Where x – the quantity of thrombocytes in 1 mkl;

a – the quantity of thrombocytes in 400 small quadrates;

200 – degree of blood dilution;400 – multiplier, which turns the result to the volume of 1 mkl (from the volume of small quadrate).

Practically the amount of thrombocytes in 400 small quadrates is multiplied by 2000 and then again by 106 and we have the amount of thrombocytes in 1 l.Answer following questions in conclusion:Is the quantity of thrombocytes normal in the examined blood?

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

Objectives: to define the total time of blood clotting and esti-mate it.Requirements for work: glass, glass hook, clock, needle, 96% ethanol, 2% alcohol solution of iodine, cotton wool.

take blood from a rat’s tail, put it on the glass and check the time.

take out the content of drop with the interval 20-30 sec (hook should be held vertically), wait, till fibrin fiber will drag after the hook.

check the time again and consider it as the moment the clotting started.

put the hook into the blood, drag the drop on the glass horizontally with the same interval. Check the time as soon as clot will be dragged after the hook, which cor-responds to the end of clotting.

Recommendations for the writing down the results:Write down the time the clotting started and the time the clot-ting ended.

165

Answer following questions in conclusion:Is the time of blood clotting normal? What does it testify?

166

Appendix I

I. Blood plasma content

Inorganic part:Fe (iron) 8,53 - 28,06 mkmol/lK (potassium) 3,8 - 5,2 mmol/lNa (sodium) 138-217 mkmol/lCa (calcium) 0,75 - 2,5 mkmol/lMg (magnesium) 0,78 – 0,91 mkmol/lP (phosphorus) 0,646 - 1,292 mkmol/lChlorides of blood 97 - 108 mkmol/lFiltrate nitrogen (not-protein) 14,28 - 25 mkmol/lUrea 3,33 - 8,32 mmol/lCreatinine 53 - 106,1 mkmol/lCreatine Men 15,25 - 45,75 mkmol/l

Women 45,75 - 76,25 mkmol/lUric acid Men 0,12 - 0,38 mkmol/l

Women 0,12 - 0,46 mkmol/lOrganic part:Total protein 65 – 85 g/lAlbumins 35 – 50 g/l

(52 – 65%)Lactatedehydrogenase (LDH) < 7 mmol (hour/l)Aldolase 0,2 – 1,2 mmol (hour/l)α-amilase (diastase of blood) 12 – 32 g/l (hour/l)Aspartateaminotransferase (AST) 0,1 – 0,45 mmol (hour/l)Alaninaminotransferase (ALT) 0,1 – 0,68 mmol (hour/l)Cholinesterase 160 – 340 mol (hour/l)Basic phosphatase 0,5 – 1,3 mmol (hour/l)Creatinkinase 0,152–0,305mmol (hour/l)Creatinphosphokinase (KPK) to 1,2 mmolLipase 0,4 – 30 mmol (hour/l)Globulins 3 – 35 g/l (35 – 48%)

167

Total bilirubin 8,5 – 20,5 mkmol/lfree bilirubin(indirect, not conjugated) 1,7 – 17,11 mkmol/lconjugated bilirubin (direct) 0,86 – 5,1 mkmol/lLipids (total amount) 5 – 7 g/lTriglicerids 0,59 – 1,77 mmol/lTotal cholesterol 2,97 – 8,79 mmol/lLipoproteins of very low density 1,5 – 2,0 g/l

(0,63 -0,69 mmol/l)low density 4,5 g/l

(3,06 – 3,14 mmol/l)high density 1,25 – 6,5 g/l

(1,13 – 1,15 mmol/l)Chylomicrons 0 – 0,5 g/l

(0 – 0,1 mmol/l)Glucose of the blood 3,3 – 5,5 mmol/lGlycolized hemoglobin 4 – 7%

II. Normal Values for Erythrocyte and LeukocyteMeasurements

Hemoglobin 13–18 g/dL (males); 12–16 g/dL (females)Hematocrit 42–52% (males); 37–48% (females)Erythrocyte count (male) 4.5–6.0 × 106/mm3 (females) 4.0–5.5 ×106/mm3Leukocyte count 5 × 103–10 × 103/mm3Differential Leukocyte CountNeutrophils 55–75%Eosinophils 2–4%Basophils 0.5–1%Lymphocytes 20–40%Monocytes 3–8%

168

Appendix II

Answers on tests for self-control:

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

Capter 21 – e, 2 – a, 3 – c, 4 – b, d, 5 – c, 6 – c, 7 – b, c, 8 – c, 9 – a, c, d, e, 10 – a, 11 – c, 12 – e, 13 – a, 14 – c, 15 – d. Chapter 31 – a, c, 2 – c, d, 3 – a, c, 4 – a, 5 – c, d, 6 – b, 7 – a, 8 – c, d, 9 – b, c, 10 – b, d, 11 – a, b, c, e, 12 – b, 13 – c, 14 – d, 15 – c.

Chapter 41 – a, b, d, e, g, 2 – b, 3 – d, e, g, 4 – a, f, 5 – d, 6 – c, 7 – a, 8 – d, 9 – e, 10 – c.

Chapter 51 – b, d, 2 – f, 3 – f, 4 – a, 5 – a, b, c, g, 6 – d, 7 – e, 8 – a, 9 – b, 10 – b, 11 – d, 12 – g, 13 – a, b, c, 14 – g, 15 – a, g.

169