1. PHYSIOLOGY OF BLOOD SYSTEM. PHYSIOLOGY OF ERYTHROCYTES.
2. RESPIRATORY PIGMENTS.
3. PHYSIOLOGY OF LEUKOCYTES.
1. Common characteristic of blood system
a) Conception of blood system (affords a pathway for the transportation of oxygen and nutrient fluids to cells that are often at a considerable distance from the heart, lungs, or digestive tract. It enables tissues to rid themselves of waste materials even though the tissues are located in the feet or hands and relatively far away from the kidneys. The blood is commonly considered to be a liquid tissue, one in which the intercellular structure is liquid rather than composed of fibers or a more or less solid substance. The circulatory system is concerned with conducting the blood through a series of tubes (arteries, veins, capillaries) to the tissues. The heart acts as a pump to supply the motive force.)
b) Blood functions (Most persons are aware that the blood carries oxygen, carbon dioxide, nutritive elements, products of metabolism, hormones and waste materials, but they are commonly unaware that the blood also has many other functions: thermoregulation, maintaining the acid-base balance of the tissues, supporting of oncotic and osmotic pressure, to form a clot, plays an important part in protecting the body from bacteria and other organism that can cause disease or other abnormal conditions.)
c) Blood volume, notion of blood reserve (The amount of blood in the body has been measured in various ways. Naturally the volume of blood can be expected to vary with the size of the body. It has been estimated at 1/20 to 1/13 of the body weight – 6- 8 %. The blood volume of a man of average size is about
d) Composition of blood, plasma (serum), quantity valuation (The liquid portion of circulating blood is called the plasma. It is a straw-colored fluid, very complex chemically, containing a wide variety of substances. The red cells, white cells, and blood platelets float in this liquid medium. Plasma: water – 92 %, solids – 8 %; inorganic chemicals: sodium, calcium, potassium, magnesium, chloride, bicarbonate, phosphate, sulfate; organic chemicals: proteins: serum albumin, serum globulin, fibrinogen; nonproteiitrogenous substances: urea, uric acid, creatine, creatinine, ammonium salts, amino acids; nonnitrogenous substances: glucose, fats, cholesterol, hormones gases: oxygen, carbon dioxide, nitrogen.)
e) Functional meaning of plasma protein (The blood plasma include such proteins as serum albumin, serum globulin, and fibrinogen. These proteins are not food proteins in the sense that they are directly absorbed from food sources or that they are food proteins being transported by the blood. Food proteins are absorbed as amino acids and transported to the tissues as such. Fibrinogen is formed in the liver, and most other plasma proteins are thought to be formed there also. Serum globulin is concerned with antibody formation – the reaction of the blood to toxins formed by bacteria or to foreign proteins introduced into the blood. Fibrinogen is essential to the clotting mechanism.)
f) Physical and chemical properties of blood (The blood proterns, present in a colloidal sol state, exert considerable osmotic pressure, up to 25 to
g) Buffer´s system of blood (Most of the tissues, including the blood, are slightly alkaline in their reaction. The pH of arterial blood is between 7,35 and 7,45. While the metabolism of the body constantly produces numerous acids and acid substances, the tissues themselves and the body fluids remain remarkably constant at a pH that is a little on the alkaline side. The principal reason for this chemical stability is the fact that the blood contains a number of alkaline substances; the chief of these is sodium bicarbonate. Weak acids produced by metabolic processes are constantly buffered by alkaline substances in the blood and in the tissues, while excess alkalinity is buffered by acids. A buffer solution contains substances that afford a reserve of alkalinity and acidity. If a weak acid or base is added to the solution, either substance is buffered by the appropriate reserve substance and a state of chemical equilibrium is maintained.)
2. Functional valuation of erythron
a) Quantity of erythrocytes, their changing (In men – 4,0-5,1•1012/L; in women – 3,7-4,7•1012/L. The quantity of erythrocytes may be increase – in pregnancy, in physical training, mental work, iewborn or decrease; and decrease – only in pathology.)
b) Function of erythrocytes (The primary function of the erythrocytes is to carry oxygen to the tissue. The erythrocytes have hemoglobin, which transport oxygen and CO2; in their membrane doing all processes that in membranes of all organs.)
c) Development of erythrocytes (Common progenitor cell – uncommited stem cell – commited stem cell – erythroblast – pronormoblast – early (basophilic) normoblast – intermidiate (polychromatorhylic) normoblast – late (eosinophilic) normoblast – reticulocyte – erythrocyte.)
d) Regulation of erythropoiesis (Coused by erythropoietins, which produced in kidneys and macrophagal system and act on red bone marrow, help to produce erythrocytes. Thiroid, epinephrine and male sex hormons increase quantity of erythrocytes and female sex hormones decrease quantity of erythrocytes.)
1. Hemoglobin (Erythrocytes derive their colour from a complex protein called hemoglobin. This substance is composed of a pigment, heme, containing iron, and the protein glohin. Hemoglobin has the power to attract oxygen molecules and to hold them in a loose chemical combination known as oxyhemoglobin. It is said, therefore, to have a chemical affinity for oxygen.)
a) Quantity and chemical structure (In man – 130-160 g/L; in woman – 120-140 g/L. It consists of four folded polypeptide chains of amino acid units. The four chains form the globin, or protein, part of the molecule. In addition there are four atoms of iron, each associated with a pigment, or heme, group of atoms. The heme group provides the red colour of the blood and also its oxygen-combining ability. The iron atoms are bivalent or in the ferrous state. It has been estimated that one erythrocyte contains approximately 280 million molecules of hemoglobin. The red, oxygen-carrying pigment in the red blood cells of vertebrates is hemoglobin, a protein with a molecular weight of 64,450. Hemoglobin is a globular molecule made up of 4 subunits. Each subunit contains a heme moiety conjugated to a polypeptide. Heme is an iron-containing porphyrin derivative. The polypeptides are referred to collectively as the globin portion of the hemoglobin molecule. There are 2 pairs of polypeptides in each hemoglobin molecule, 2 of the subunits containing one type of polypeptide and 2 containing another. Iormal adult human hemoglobin (hemoglobin A), the 2 types of polypeptide are called the a. chains, each of which contains 141 amino acid residues, and the b chains, each of which contains 146 amino acid residues. Thus, hemoglobin A is designated a2b2.)
b) Methods of definite (gasometric – definition of gases, such as O2, colorimetric – definition of colour substances, ironmetric – definition of iron concentration.)
c) Combination of hemoglobin, their peculiarities (As the blood passes through a capillary network in the thin air sacs of the lungs, oxygen enters into a loose chemical combination with hemoglobin (oxyhemoglobin) and is carried to the tissues. There, as the blood passes through tissue capillaries, the hemoglobin loses oxygen to the tissues and is then referred to as reduced hemoglobin. Arterial blood, after passing through the lungs, is a somewhat brighter red than that found in the veins, but venous blood is never blue. The blue colour of veins close to the surface is due to the absorption of red and yellow rays of light and the reflection of blue and green light. Erythrocytes not only function to carry oxygen to the tissues, but indirectly they also function in carrying carbon dioxide away from the tissues. ). When blood is exposed to various drugs and other oxidising agents in vitro or in vivo, the ferrous iron (Fe2+) in the molecule is converted to ferric iron (Fe3+), forming methemoglobin. Methemoglobin is dark-coloured, and when it is present in large quantities in the circulation, it causes a dusky discoloration of the skin resembling cyanosis.)
d) Exchange of iron in the organism (In the blood-destroying organs, the hemoglobin breaks down into an iron-free and the iron-bearing portions. The latter is decomposed into bilirubin and an iron compound. Both are carried to the liver, where the bilirubin is excreted in the bile as one of the bile pigments, while the iron, if not needed for the formation of new red blood cells, is stored. Other way entering of iron is the food.)
e) Destruction of erythrocytes and hemoglobin (Erythrocytes can live only a limited time. Estimated of the amount of bile pigment excreted daily indicate that in a normal adult approximately 20000000 red blood cells are destroyed every minute. The life of red blood cells are nearly 120 days. Blood cells are lost by the processes of hemolysis and fragmentation, which occur throughout the circulatory system, and phagocytosis of whole cells and cell fragments, which takes place in the cells of the reticuloendothelian tissues, especially those in the spleen, the liver, and the bone marrow.)
f) Notion of colouring index, average content of hemoglobin in erythrocytes (Colouring index and average content of hemoglobin in erythrocytes shows the degree of erythrocytes filling by hemoglobin. Norm index of colouring index defenition is 0,85-1,05; norm average content of hemoglobin in erythrocytes is 26-33 picogramm.)
2. Myoglobin
a) Localisation, structure (Hem is also part of the structure of myoglobin, an oxygen-binding pigment found in red (slow) muscles and in the respiratory enzyme cytochrome c. Porphyrins other than that found in hem play a role in the pathogenesis of a number of metabolic diseases (congenital and acquired porphyria, etc.))
b) Functional peculiarities (It may be the reserve pigments, which give the tissue oxygen in a small oxygen condition.)
1. Influence of plasma composition on blood reology (Reology is a strong friction which arise in the case of fluids’ layers moving with the different speed.)
a) Protein and nonprotein components (Depends of molecular weight of protein).
b) Physical and chemical factors (Depends of pH of blood, temperature.).
2. Deformation properties of erythrocytes (Erythrocytes may pass through capillars, which have a smaller diameter than erythrocytes.)
a) Membrane mechanisms (Depends of structure of membrane, it flexible.)
b) Role of erythrocytes content (Depends of structure of hemoglobin.)
3. Physiological analysis of erythrocytes sedimentation speed (ESS) (In a blood which caot clotting erythrocytes sedimentation. Iorm erythrocytes sedimentation speed in male is 2-10 mm/hour, in female is 2-15 mm/hour. It necessary for diagnose the inflammatory processes in human organism.)
a) Notion about erythrocytes aggregation (In healthy person erythrocytes aggregation is absent. Erythrocytes aggregation is develop of connection between erythrocytes (small bridges) and forming chains.)
b) Influence of blood components on erythrocytes sedimentation speed (It may be 2 groups of factors – erythrocytes and plasma. Erythrocytes sedimentation speed is increase in a case of decrease the quantity of erythrocytes, decrease pH of blood, quantity of albumins. Erythrocytes sedimentation speed is increase in a case of increase the quantity of hemoglobin, volume of erythrocytes, protein – fibrinogen, cholesterol, gamma-globulins, increase pH of blood.)
c) Influence of physiological condition on erythrocytes sedimentation speed (Erythrocytes sedimentation speed increase iewborn, pregnancy women, in a case of different inflammation processes.)
4. Common characteristic of erythrocytes resistance
a) Determine the notion “resistance” and “hemolysis” (Resistancy is a property of erythrocytes to be hole in solution with different concentration. Hemolysis is a process of going out of hemoglobin from erythrocytes in plasma. It may be in cases of destroyed the erythrocytes membranes and without destroyed the erythrocytes. Level of osmotic resistance of erythrocytes is the concentration of NaCl in solution in which hemolyse erythrocytes. Minimal resistance of erythrocytes (0,50-0,45 % of NaCl) – in this concentration of NaCl destroyed erythrocytes with the smallest resistance. Maximal resistance of erythrocytes (0,34-0,32 % of NaCl) – in this concentration of NaCl destroyed all erythrocytes. Osmotic resistance of erythrocytes may decrease and hemolysis may be in a higher concentration of NaCl.)
b) Kinds of hemolysis (There are 3 kinds of hemolysis: biological, chemical, physical. Biological causes of hemolysis are poisons of snakes, for example; chemical – strong acids, ether, for example; mechanical – in patient with the pathology of membranes in a case of running, jumping, for example; temperature – higher temperature.)
Key words and phrases: oncotic and osmolic pressure, regulatory influences; erythron; alkalinity and acidity; buffer; plasma; serum; common progenitor cell; uncommited stem cell; commited stem cell; erythroblast; early (basophilic) normoblast; intermidiate (polychromatorhylic) normoblast – late (eosinophilic) normoblast; reticulocyte; erythropoietins, hemoglobin, myoglobin, coloring index, average content of hemoglobin in erythrocytes, iron, reticuloendothelian tissues, pigment, bilirubin, heme, porphyrin, cytochrome c, oxygen-binding, phagocytosis, iron-containing, iron-bearing, iron-free, erythrocytes sedimentation speed, pipettes, water bath, centrifuge, reology, resistancy, hemolysis, hematocritic index, hematocrit
1. Serum consist of:
a) Albumins, globulins, fibrinogen; b) Erythrocytes, platelets, proteins; c) Plasma without fibrinogen; d) Erythrocytes, albumins, globulins; e) Globulins, fibrinogen, water
2. The main function of blood is:
a) Tranpsort of different substances; b) Maintaining the acid-base balance; c) To form a clot; d) Protective; e) Supporting of oncotic and osmolic pressure
3. Such cells ripened only in bone marrow:
a) Uncommited stem cell, reticulocyte; b) Common progenitor cell, erythroblast;
c) Commited stem cell, late normoblast; d) Intermidiate and eosynophylic normoblast; e) Early normoblast, erythrocyte
4. The normal quantity of erythrocytes in man is:
a) 3,4•1012/L; b) 3,8•1012/L; c) 4,3•1012/L; d) 5,2•1012/L; e) 6,0•1012/L
5. The normal quantity of hemoglobin in health man is:
a) 105 g/L; b) 115 g/l; c) 125 g/L; d) 135 g/L; e) 165 g/L
6. The erythrocytes destroyed in:
a) Liver, lungs, brain, spleen; b) Liver, spleen, bones, lungs; c) Liver, spleen, blood bed, lungs; d) Spleen, brain, blood bed, liver; e) Lungs, spleen, bones, brain
7. The origin (best of all) of iron is:
a) Meat of animal, coffee, fruits; b) Liver of animal, fruits, vegetables; c) Fruits, vegetables, tea; d) Vegetables, meat, coffee; e) Tea, coffee, vegetables
8. The normal quantity of hemoglobin in health women is:
a) 95 g/L; b) 105 g/L; c) 115 g/L; d) 125 g/L; e) 145 g/L
9. The best, most modern method of hemoglobinsdefinition is:
a) By means of Sally’s hemometer; b) By means of photoelectrohemometer; c) Cyanmethemoglobin method
10. What is the males’ erythrocytes sedimentation speed iorm?
a) 2-10 mm/hour; b) 2-15 mm/hour; c) 4-9 mm/hour; d) 5-10 mm/hour; e) 4-5 mm/hour
11. What is the males’ erythrocytes sedimentation speed iorm?
a) 2-10 mm/hour; b) 2-15 mm/hour; c) 4-9 mm/hour; d) 5-10 mm/hour; e) 4-5 mm/hour
12. What is the viscosity of blood iorm?
a) 1,9-2,1; b) 3; c) 5; d) 4-9; e) 5-10
13. What is the hematocrit of blood of female iorm?
a) 0,20-0,55 L/L; b) 0,30-0,55 L/L; c) 0,36-0,42 L/L; d) 0,40-0,48 L/L; e) 0,55-0,60 L/L
14. What is the hematocrit of blood of male iorm?
a) 0,20-0,55 L/L; b) 0,30-0,55 L/L; c) 0,36-0,42 L/L; d) 0,40-0,48 L/L; e) 0,55-0,60 L/L
15. What is the density of blood iorm?
a) 1,025-1,034 g/sm3; b) 1,055-1,060 g/sm3; c) 1,060-1,080 g/sm3; d) 1,075-1,084 g/sm3; e) 1,090-1,098 g/sm3
16. What is the density of plasma iorm?
a) 1,025-1,034 g/sm3; b) 1,055-1,060 g/sm3; c) 1,060-1,080 g/sm3; d) 1,075-1,084 g/sm3; e) 1,090-1,098 g/sm3
17. What is the density of erythrocytes iorm?
a) 1,025-1,034 g/sm3; b) 1,055-1,060 g/sm3; c) 1,060-1,080 g/sm3; d) 1,075-1,084 g/sm3; e) 1,090-1,098 g/sm3
Real-life situations to be solved:
1. Patient N., 55 years old, was hospitalized in a heamatologic unit. Doctor diagnosed anemia. What may be the cause of the disease?
2. Patient A., 45 years old, was hospitalized in a surgery unit with the complaints of the common tiregness, pain in abdomen, dizziness. What is the cause of the disease, if you know that he had the stress on the ever?
3. Patient N., 20 years old, was hospitalised in a neurology unit. Analyses results are quantity of erythrocytes – 3,9•1012/L, hemoglobin – 126 g/L. To definite colouring index, average content of hemoglobin in erythrocytes.
4. Patient A., 45 years old, was hospitalised in a cardiac unit. In his analyses quantity of carboxyhemoglobin is 8 %. What is the cause of the disease?
5. Patient F. has the follow analyses: erythrocytes – 4,6 T/L, hemoglobin – 136 g/L, leukocytes – 9,0 G/L, erythrocytes sedimentation speed – 9 mm/hour. Is this results iorm?
6. Patient C. has the follow analyses: erythrocytes – 3,8 T/L, hemoglobin – 126 g/L, leukocytes – 5,0 G/L, erythrocytes sedimentation speed – 12 mm/hour. Is this results iorm?
7. Patient C. has the follow analyses: viscosity of blood – 5,0; density – 1,058 g/sm3; hematocrit – 0,42 L/L; erythrocytes sedimentation speed – 9 mm/hour. Is this results iorm?
8. Minimal resistancy of blood is 0,60 % solution of NaCl, maximal resistancy of blood is 0,40 % solution of NaCl. What do You can say about this blood? Is this at norm?
Answers for the Self-Control
1. c;
1 real-life situation – There may be decreasing of erythrocytes – blooding from different organs, cancer.
2 real-life situation – There may be blooding from the stomach
3 real-life situation – Colouring index – 0,97; average content of hemoglobin in erythrocytes – 32 pg. It is the physiological norm.
4 real-life situation – This man is the smoker.
5 real-life situation – Yes, this results is at norm for male (erythrocytes in male – 4,0-5,1 T/L, hemoglobin in male – 130-160 g/L, leukocytes – 4,0-9,0 G/L, erythrocytes sedimentation speed in male – 2-10 mm/hour).
6 real-life situation – Yes, this results is at norm for female (erythrocytes in female – 3,7-4,7 T/L, hemoglobin in female – 120-140 g/L, leukocytes – 4,0-9,0 G/L, erythrocytes sedimentation speed in female – 2-15 mm/hour).
7 real-life situation – Yes, this results is at norm (norm viscosity of blood – 5,0; density – 1,055-1,060 g/sm3; hematocrit – 0,36-0,42 L/L in female and 0,40-0,48 L/L in male; erythrocytes sedimentation speed – 2-15 mm/hour in female and 2-10 mm/hour in male).
8 real-life situation – Minimal resistancy of fresh blood – 0,50-0,45 % solution of NaCl, maximal resistancy of fresh blood – 0,34-0,32 % solution of NaCl. Minimal resistancy of unclotting blood – 0,70-0,60 % solution of NaCl, maximal resistancy of fresh blood – 0,40-0,15 % solution of NaCl. That is why this the unclotting blood.
Estimation of erythrocytes in Horyaev’s camera
Full the mixer (melangeur) for erythrocytes with blood to the sign 0,5. Full it to the sign 101 with hypertonic solution (3 % NaCl).
If you want to solute in 200 times you can do this even without mixer. Full dry and clean tube with 4 ml of NaCl solution and put 0,02 ml of blood.
Put tegmental glass on prominent part of clean and dry glass with Goryaev’s net. Then move covering glass in sagittal direction. The appearance of color rings in the places of contact of glass surfaces will prove it. Height of creating camera over Horyaev’s net will be 0,1 ml.
Release dissolutions from the capillary on cotton wool and fill the camera with blood solution from the extension. If dilution was in tube, than use Paster’s papper or other capilary to take the mixture and fill camera.
While doing it, please, look after the forming of small vesicles. In 1-2 minutes after segmentation of erythrocytes estimate their quantity in 5 large squares, divided in 16 small squares (in small extension of microscope in dark field vision). Estimation must be carried out from the upper square than down in diagonal direction. The quantity of erythrocytes in
X=[(a•200)/80]•4•109, where
x – quantity of erythrocytes in
200 – dilution of blood; 80 – quantity of small squares in 5 large ones.
Make the scheme of Horyaev’s net with erythrocytes over it – in test paper.
In conclusion define wheather the quantity of erythrocytes is within physiology norm. If it is not, than explain the reason of this phenomena.
Definition of hemoglobin by means of Sally’s hemometer
To fill the middle, graduated test-tube of hemometer by means of pipette to the lower sigh with HCl. Put 0,02 ml of blood their carefully. Mix the content of tube and leave it for 5 minutes in order hemoglobin converted into chloride hematin. Then using distillated water making the colour of the standard. Compare the colour in passing light, holding the hemometer on the eyes-level. According to the mark on the tube find the quantity of hemoglobin and turn it into the SI system. Make a schema of Sally’s hemometer in your test-papper.
Please, indicate in conclusion whether the result is within physiological standards. If it is not, explain please the reason.
Definition of hemoglobin using cyanmethemoglobin method.
Fill the tube with 5 ml of transformed solution and 0,02 ml of investigating blood. Then find apt density of this mixture and standard solution by means of photoelectrocolorymeter (the length of wave is 500-600 nm, green light filter). Find the content of hemoglobin according the formula:
Hb g/L=( E investigating test : E standard test ) • 251 • C, where
E investigating test and E standard test – optic density;
C – concentration of Hb in standard solution – 0,5975 g/L;
251 – coefficient of blood dilution.
Indicate please in conclusion whether the result is withing physiological standards. If not, explain the reason of phenomena.
Definition of colouring index, average content of hemoglobin in erythrocytes.
Colouring index (CI) find according the formula:
CI=( 3 • K1 ) /
K1 – quantity of hemoglobin
Average content of hemoglobin (ACH) in one erythrocyte find according the formula:
ACH=K1 /
K1 – quantity of hemoglobin
Indicate please in conclusion whether the result is withing physiological standards. If not, explain the reason of this phenomenon.
Determination of haematocritis index.
Haematocritis is cleaned by sodium ocsalat solution is filled up by the blood. To strain the rubber ring on the capillar. To centrifugate during 30 minutes by 3000 turns. To note the volume of erythrocytes and express it in international units. Is this hematocritic index norm?
If it is not, what is the cause of deviation?
Determination of erythrocytes sedimentation speed (ESS).
Panchenkov’s capillar is filled up of sodium citrate solution to ”P” level (solution–розчин). To empty it in small cup. Then the blood is picked to “К” level (blood–кров) twice. To pour out of the blood to sodium citrate, than mix. The capillar is filled up of mixture to level “K” and stay in vertical state in Panchenkov’s apparatus.
To determinate ESS by means of formula:
ESS=42-(7,5 x quantity of erythrocytes in 1L)=mm/hour.
Where 42 and 7,5-coefficients.
Compare appropriate quantity ESS with real.
If it is not norm, what is the cause of deviation?
1. Physiological valuation of white blood (There are two major groups of white cells: the first group includes those cells that have granules in the cytoplasm and possess a nucleus of two or three lobes. They are called granular, or poly-morphonuclear, leukocytes. The second group includes those cells that do not have granular in the cytoplasm and in which the nucleus is more or less spherical in shape. These are the nongeanular white cells; the grouping includes lymphocytes and monocytes.)
a) Common function of white cells (Phagocytosis refers to the ability of neutrophils to ingest bacteria or other foreign bodies. They contain protein-digesting enzymes that enable them to digest most of the materials they engulf. There is a rise in the number of eosinophils in some cases of allergy, possibly in response to toxic substances released by the allergic reaction. Basophils in the blood are said to contain histamine and a heparinlike substance. Histamine dilates capillaries and often permits fluid to move through the capillary wall into the tissues: heparin is an anticoaguiant of the blood. Apparently tissue basophils become the mast cells of the tissues. The large granules of mast cells are thought to store enzymes.Some kinds of leukocytes, especially neutrophils and monocytes, exhibit ameboid movement and are actively phagocytic.)
b) Quantity of leukocytes and their changes (White cells are nucleated and somewhat variable in size and shape. Their numbering is 4-9•109 per liter. The number of lymphocytes are – 18-37 %, monocytes – 3-11 %, eosinophils –0,5-5 %, basophils – 0-1 %, juvenile neutrophile – 0-1 %, relating to stab (rod-shaped) neutrophil – 1-6 %, segmented neutrophil – 47-72 %. The number of leukocytes and different kind of leukocytes may increase. This condition called – leukocytosis, lymphocytosis, monocytosis, neutrophilosis, eosinophilia, basophilia. The number of leukocytes and different kind of leukocytes may decrease. This conditioamed – leukopenia, lymphocytopenia, monocytopenia, neutropenia, eosinopenia.)
c) Leukopoiesis (Development of monocytes: common progenitor cell – uncommited stem cell – commited stem cell – monoblast – promonocyte – monocyte – tissue macrophage. Development of lymphocytes: common progenitor cell – bone marrow lymphocytes precursor – lymphoblast – prolymphocyte – large lymphocyte – small lymphocyte. Development of gtanulocytes: common progenitor cell – uncommited stem cell – commited stem cell – myeloblast (basophil, neutrophil, eosinophil) – promyelocyte – myelocyte – metamyelocyte – juvenile – rod-shaped neutrophil (basophil, eosinophil), segmented neutrophil, basophil, eosinophil. Lymphocytes in the fetus are thought to arise first in the thymus. Later they are found in lymph nodes, spleen, and other lymphoid tissues as well as in bone marrow.)
2. Significance of leukocytes in immunity security (The small lymphocyte has been called the immunologically competent cell. It appears that thymus is the essential structure capable of establishing an immunologic response in lymphoid precursor cells or in thymic lymphocytes. There may be a The small lymphocyte has been called the immunologically competent cell. It appears that in the fetus the thymus is the thymic humoral factor, possibly a hormone, that influences the development of lymphocytes in the spleen and lymph nodes. These lymphocytes then become capable of multiplying and producing an immune response. Monocytes are actively motile and phagocytic. It is thought that they function in contributing to the repair and reorganization of tissues. Monocytes and macrophages are capable of engulfing old, wornout neutrophils, mast cells, antigens, and particles of tissue in the process of cleaning up area of inflammation or infection after the initial stages have been passed and recovery is in progress.)
a) Physiological role of T-lymphocytes (There are receptors to antigens on the membrane of T-lymphocytes, which helps to distinguish genetic heterologous substances.)
b) Functional significance of B-lymphocytes (B-lymphocytes syntheses the immunoglobulins such as IgM, IgN, IgA, IgG, IgB, IgE.)
System of mononucleares phagocytes (These is the system, which common the cells with one nucleus, common origin from red bone marrow, common function of high specific phagocytosis.)
a) Origin of mononucleares phagocytes system (It origin from promonocytes of red bone marrow. Tissue monocytes named macrophage. They enter the circulation from the bone marrow, but after about 24 hours they enter the tissues to become tissue macrophages. All of the tissue macrophages come from circulating monocytes. The tissue macrophage system has generally been called the reticuloendothelial system.)
b) Spreading in the human organism (There are many macrophages in different organs. The macrophage of lungs named alveolar macrophages of lungs, the macrophage of spleeamed free or fixed macrophages of spleen, the macrophage of liver named Kupffer cells, the macrophage of skiamed Langergans cells, the macrophage of braiamed microglia etc.)
c) Functions of mononucleares phagocytes system (It has the function of high specific phagocytosis. Its takes please in synthesis of antibodies, in reactions of cells immunity, such as rejection of transplant (graft), protection of tumor cells. The monocytes, like neutrophilic leukocytes, are actively phagocytic and contain peroxidase and lysosomal enzymes. The macrophages migrate in response to chemotactic stimuli and engulf and kill bacteria by processes that are generally similar to those occurring ieutrophils. They also play a key role in cellular immunity because proliferation of T-lymphocytes requires that they come in physical contact with macrophages that have taken up and processed antigen.)
LEUKOCYTES FORMULA
1. Common characteristic of the leukocytes formula
a) Kind of leukocytes, bases of it separation (There are two major groups of white cells: the first group includes those cells that have granules in the cytoplasm and possess a nucleus of two or three lobes. They are called granular, or poly-morphonuclear, leukocytes. The second group includes those cells that do not have granulea in the cytoplasm and in which the nucleus is more or less spherical in shape. These are the nongeanular white cells; the grouping includes lymphocytes and monocytes. Of these, the granulocytes are the most numerous. Young granulocytes have horseshoe-shaped nuclei that become multilobed as the cells grow older. Most of them contaieutrophilic granules (neutrophils), but a few contain granules that stain with acid dyes (eosinophils), and some have basophilic granules (basophils). The other 2 cell types found normally in peripheral blood are lymphocytes, cells with large, round nuclei and scanty cytoplasm; and monocytes, cells with abundant agranular cytoplasm and kidney-shaped nuclei. Acting together, these cells provide the body with powerful defenses against tumors and viral, bacterial, and parasitic infections.)
b) Definition of “leukocytes formula‘s” notion and its quantitative expression (The number of different kinds of leukocytes in the volume of blood called “leukocytes formula”. It quantitative determination in 109/L. We has determination the number of leukocytes in 100 leukocytes of the smear, that‘s way the quantity of leukocytes may be occur in percent. The number of lymphocytes are – 0,18-0,37 (18-37 %), monocytes – 0,03-0,11 (3-11 %), eosinophils –0,005-0,05 (0,5-5 %), basophils – 0-0,01 (0-1 %), juvenile neutrophile – 0-0,01 (0-1 %), relating to stab (rod-shaped) neutrophil – 0,01-0,06 (1-6 %), segmented neutrophil – 0,47-0,72 (47-72 %).)
1. erythrocytes 2. lymphocytes 3. monocyte 4. neutrophil 5. eosinpphil 6. basophil 7. platelets
c) The index of nuclear’s changing of neutrophils, it interpretation (The number of circulating granulocytes is regulated with precision. In health, the number is held at a constant level; when infection occurs, the number in the blood rises dramatically and rapidly. The release of granulocytes from the bone marrow appears to be stimulated by granulocyte-releasing factors in the blood. One or more additional factors (granulopoietins) stimulate the conversion of committed stem cells to granulocytes. The nuclear’s changing of neutrophils is increase when the number of young neutrophils increase and decrease when the number of segment neutrophils are increase.)
2. Functional peculiarities of leukocytes (All granulocytes contain the enzyme myeloperoxidase. This enzyme, which has a molecular weight of about 150,000, catalyzes the formation of hypohalite ions that aid in killing ingested bacteria.)
a) Neutrophils (The neutrophils seek out, ingest, and kill bacteria and have been called the body‘s first line of defense against bacterial infections. The average half-life of a neutrophil in the circulation is 6 hours. The nucleus appears to vary with age. The younger mature cells have a bilobed or trilobed nucleus with thick connections between the lobes, whereas in older cells there are more lobes in the nucleus and the connections between the lobes are thin. The granules of the cytoplasm are very fine, and when stained with Wright‘s or some similar stain, the granules take both acid and basic stains and appear as a lavender or lilac color. Many of the neutrophils enter the tissues; they first adhere to the endothelium and then insinuate themselves through the walls of the capillaries between endothelial cells by a process called diapedesis. Many of those that leave the circulation enter the gastrointestinal tract and are lost from the body. When bacteria invade the body, the bone marrow is stimulated to produce and release large numbers of neutrophils. Phagocytosis refers to the ability of neutrophils to ingest bacteria or other foreign bodies. They contain protein-digesting enzymes. Their phagocytic activity is important in helping to rid the body of injurious bacteria.)
b) Eosinophils (Resembling neutrophils, eosinophils are slightly larger, and the nucleus is usually bilobed. The granules in the cytoplasm are larger and stain a bright red with acid dyes such as eosin. These cells make up only 0,5 to 5 percent of the total number of white cells in the blood, but in the tissues they can congregate in considerable numbers. According to some workers, eosinophils are considered to be less active and not so highly phagocytic as neutroohils. There is a rise in the number of eosinophils in some cases of allergy, possibly in response to toxic substances released by the allergic reaction. The eosinophils attack some parasites, and they inactivate mediators released from mast cells during allergic reactions. The circulating eosinophil level is often elevated in patients with allergic diseases.)
c) Basophils (Basophils have a bilobed nucleus, and the large cytoplasmic granules stain a deep blue with basic stains such as methylene blue or with Wright’s stain, which contains methylene blue. Basophils constitute only 0,5 percent of the white cells of the blood. Not much is known about the motility less active or phagocytic activity of basophils, but they are considered to be than eosinophils. Basophils in the blood are said to contain histamine and a heparinlike substance. Histamine dilates capillaries and often permits fluid to move through the capillary wall into the tissues: heparin is an anticoaguiant of the blood. Apparently tissue basophils become the mast cells of the tissues. The large granules of mast cells are thought to store enzymes. The basophils contain histamine and heparin, but their role in the maintenance of normal balance between the clotting and anticlotting systems is uncertain.)
Key words and phrases:
smear, fat-free (degreased) glass, grinding border, Romanovsky’s stain, grinding, vinegar acid, Goryaev’s net, mononucleares phagocytes system, immunity security, T, B-lymphocytes, granulocytes, agranulocytes, neutrophils, eosinophils, basophils, lymphocytes, monocytes, juvenile, segmented, myeloblast, promyeloblast, myelocytes: basophyl, neutrophil, eosinophil, Kupffer cells, microglia, Langergans cells, transplant (graft), tumor, distinguish, heterologous; granulocytes, agranulocytes, polymorphonuclear leukocytes, young granulocytes, horseshoe-shaped nuclei, multilobe, neutrophilic granules, neutrophils, acid dyes, eosinophils, basophilic granules, basophils, peripheral blood, lymphocytes, scanty cytoplasm, monocytes, abundant agranular cytoplasm, kidney-shaped nuclei, powerful defenses, tumors, viral, bacterial, parasitic infections, smear, phagocytes system, juvenile, segmented, myelocytes, clotting and anticlotting systems.
1. The macrophages origin is:
a) Granulocytes; b) Basophyls, eosinophils; c) Promonocytes; d) Uncommited stem cell; e) Monoblast
2. Leukocytosis is:
a) Increase of leukocytes quantity above 4,0•109/L; b) Increase of leukocytes quantity above 9,0•109/L; c) Decrease of leukocytes quantity lower 4,0•109/L; d) Decrease of leukocytes quantity lower 9,0•109/L; e) Quantity of leukocytes is (4,0-9,0)•109/L
3. Leukopenia is:
a) Increase of leucocytes quantity above 4,0•109/L; b) Increase of leucocytes quantity above 9,0•109/L; c) Decrease of leucocytes quantity lower 4,0•109/L; d) Decrease of leucocytes quantity lower 9,0•109/L; e) Quantity of leukocytes is (4,0-9,0)•109/L
4. Which kind of cells takes place in immunologic reaction?
a) B-lymphocytes, T-lymphocytes, monocytes; b) B-lymphocytes, neutrophil, monocytes; c) B-lymphocytes, macrophages, plasmocytes; d) Plasmocytes, granulocytes, monocytes; e) Basophils, eosinophils, monocytes
5. The increase of the index of nuclear’s changing of neutrophils may be:
a) Pneumonia; b) Headache; c) Toothache; d) Anemia; e) Climacteric period
6. The index of nuclear’s changing of neutrophils index is 0,09, when :
a) The quantity of young neutrophils is 1 %, stab neutrophils – 6 %, segment neutrophils – 52 %; b) The quantity of young neutrophils is 1 %, stab neutrophils – 7 %, segment neutrophils – 43 %; c) The quantity of stab neutrophils is 4 %, segment neutrophils – 70 %; d) The quantity of stab neutrophils is 7 %, segment neutrophils – 49 %; e) The quantity of young neutrophils is 1 %, stab neutrophils – 5 %, segment neutrophils – 67 %
7. What are the largest cells in the blood?
a) Lymphocytes; b) Basophils; c) Eosynophils; d) juvenile neutrophils; e) stab neutrophils
8. Which kind of cells takes place clotting reaction?
a) Basophil; b) Lymphocyte; c) Monocyte; d) Eosinophil; e) Neutrophil
1. Patient S., 36 years old, was examinated after great physical activity. In his blood the quantity of leukocytes was 9,2•109/L, volume of blood – 9 %. What is the mechanism of this change?
2. Patient C., 25 years old, was hospitalized in a obstetrics unit. What is the quantity of leukocytes in her analyzers? What is it called?
3. Patient H., 39 years old, was examinated in therapeutic unit. In his blood the quantity of leukocytes was 5,6•109/L. In leukocytes formula the number of neutrophils was – stab neutrophils – 0,18•109/L, segment neutrophils – 2,94•109/L, basophils – 0,06•109/L, eosiniphils – 0,06•109/L, lymphocytes – 2,16•109/L, monocytes – 0,60•109/L. Please, analyze the results. Definit the index of nuclear’s changing of neutrophils, please.
4. Patient C., 25 years old, was examinated in clinical laboratory. In common analyze of blood the quantity of cells was erythrocytes – 3,70•1012/L, leukocytes –8,00•109/L, thrombocytes – 182•109/L; hemoglobin – 125 g/L. In leukocytes formula the number of neutrophils was – young 0,12•109/L, stab neutrophils – 0,60•109/L, segment neutrophils – 2,82•109/L, basophils – 0,06•109/L, eosiniphils – 0,36•109/L, lymphocytes – 1,44•109/L, monocytes – 0,60•109/L. What is the change of leukocytes quantity in her analyzers? What is it normal quantity?
Answers for the Self-Control
1. c; 2. b; 3. c;
1 real-life situation – Increase of blood volume coused by throw it away from depo, leukocytoses – stimulated of leukopoiesis by leukopoiethins
2 real-life situation – It increased. It called – leukocytoses. It may be after meal, emotion
3 real-life situation – The quantity of leukocytes is normal (4-9•109/L). In leukocytes formula the number of neutrophils – young – norma (0-0,06•109/L), stab neutrophils – norma (0,06-0,36•109/L), segment neutrophils – norma (2,82-4,32•109/L), basophils – norma (0-0,06•109/L), eosiniphils – norma (0,03-0,30•109/L), lymphocytes – norma (1,08-2,22•109/L), monocytes – norma (0,60-0,09•109/L). Iindex of nuclear’s changing of neutrophils=(0+0+0,18):2,94=0,06 (normal number – 0,06-0,09).
Increase of blood volume coused by throw it away from depo, leukocytoses – stimulated of leukopoiesis by leukopoiethins
4 real-life situation – The quantity of erythrocytes is decrease (norma – 4,0-5,1•1012/L), leukocytes is normal (norma – 4-9•109/L), thrombocytes is normal (norma – 180-320•109/L); the concentration of hemoglobin is decreased (norma – 130-160 g/L). In leukocytes formula the number of neutrophils – young – increase (norma – 0-0,06•109/L), stab neutrophils – increase (0,06-0,36•109/L), segment neutrophils – norma (2,82-4,32•109/L), basophils – norma (0-0,06•109/L), eosiniphils – increase (norma – 0,03-0,30•109/L), lymphocytes – norma (1,08-2,22•109/L), monocytes – norma (0,60-0,09•109/L).
Preparation of blood smear
Make please blood smear on the fat-free glass stage without grinding borders. In order to make it, please put the drop of blood on the right part at the distance about 1-1,5 sm from short rib. Take in right hand glass stage with grinding borders and push it on the left to the drop of blood in order to locate it in angle of 45. When the drop of blood extent on the left along the degreased glass, stop at the distance about 1 sm to the border.
Dry this smear, fix (about 20 min) and draw it 45 min by means of Romanovsky’s stain. After it you must wash it and dry.
Indicate, please, in conclusion whether it was made according to the established rule.
Estimation of general quantity of leukocytes
Fill the mixture for leukocytes to the sign 0,5 with blood and to the sign 11 with solution of vinegar acid with gentianviolette. Sometime it can be made in a tube. Put 0,4 ml of the vinegar acid solution with gentianviolette and 0,02 ml of blood into dry, clean test-tube. Fill Goryaev’s camera with this mixture, just like during the estimation of erythrocytes.
Estimate the leukocytes in 100 large squares, united into groups (every of it consists of 4 big squares) in dark field of vision in small extension of microscope.
Find the quantity of leukocytes according to the formula:
X=[(A•20) / 1600] • 4 • 109, where
X – quantity of leukocytes in 1liter.
a – quantity of leukocytes in 100 large squares.
20 – dilution of blood.
1600 – quantity of small squares in 100 large ones.
Make a scheme of Goryaev’s net with leukocytes over it in your note-book. Indicate please in conclusion whether the estimated quantity of leukocytes is within physiological standards. If not, explain the reason of phenomena.
Estimation of leukocytes formula.
Find 100 leukocytes in the smear, made on the fast lesson, under immersion, moving the field of vision along zigzag line (line of Meander). According to these results, find correlation between different kinds of leukocytes. Make a table and a scheme of the found leukocytes.
|
Units |
Neutrophils |
Baso-phils |
Eosino-phils |
Limpho-cytes |
Mono-cytes |
||
|
juve-nile |
Stab neutro-phils |
Segmented neutrophils |
|||||
|
relative |
|
|
|
|
|
|
|
|
absolute |
|
|
|
|
|
|
|
Indicate please in conclusion whether this leukocyte formula is within physiological standards.
Definition of the index of nuclear’s changing of neutrophils.
This index can be found out according to the formula:
NCN=(M+J+S1)/S2, where
M – myelocytes, J– juvenile, S1 – stab neutrophils, S2 – segmented neutrophils.
Indicate please in conclusion whether this formula, index is within physiological standards. If not, find the reason of these phenomena.
Stem Cells, Growth Factors, & Differentiation
Stem cells are pluripotential cells capable of self-renewal.
Pluripotential Hematopoietic Stem Cells
It is believed that all blood cells arise from a single type of stem cell in the bone marrow. Because this cell can produce all blood cell types, it is called a pluripotential stem cell. These cells proliferate and form one cell lineage that will become lymphocytes (lymphoid cells) and another lineage that will form the myeloid cells that develop in bone marrow (granulocytes, monocytes, erythrocytes, and megakaryocytes). Early in their development, lymphoid cells migrate from the bone marrow to the thymus, lymph nodes, spleen, and other lymphoid structures, where they proliferate Progenitor & Precursor Cells.
Stem Cells, Growth Factors, & Differentiation
Stem cells are pluripotential cells capable of self-renewal.
Pluripotential Hematopoietic Stem Cells
It is believed that all blood cells arise from a single type of stem cell in the bone marrow. Because this cell can produce all blood cell types, it is called a pluripotential stem cell. These cells proliferate and form one cell lineage that will become lymphocytes (lymphoid cells) and another lineage that will form the myeloid cells that develop in bone marrow (granulocytes, monocytes, erythrocytes, and megakaryocytes). Early in their development, lymphoid cells migrate from the bone marrow to the thymus, lymph nodes, spleen, and other lymphoid structures, where they proliferate Progenitor & Precursor Cells.
Hematopoiesis is therefore the result of simultaneous, continuous proliferation and differentiation of cells derived from stem cells whose potentiality is reduced as differentiation progresses. This process can be observed in both in vivo and in vitro studies, in which colonies of cells derived from stem cells with various potentialities appear. Colonies derived from a myeloid stem cell can produce erythrocytes, granulocytes, monocytes, and megakaryocytes, all in the same colony.
In these experiments, however, some colonies produce only red blood cells (erythrocytes). Other colonies produce granulocytes and monocytes. Cells forming colonies are called colony-forming cells (CFC) or colony-forming units (CFU). The convention iaming these various cell colonies is to use the initial letter of the cell each colony produces. Thus, MCFC denotes a monocyte-forming colony, ECFC forms erythrocytes, MGCFC forms monocytes and granulocytes, and so on.
Hematopoiesis depends on:
1-Favorable micro-environmental conditions and
2-The presence of growth factors.
Once the necessary environmental conditions are present, the development of blood cells depends on factors that affect cell proliferation and differentiation. These substances are called growth factors, colony-stimulating factors (CSF), or hematopoietins (poietins).
Bone Marrow
Under normal conditions, the production of blood cells by the bone marrow is adjusted to the body’s needs, increasing its activity several-fold in a very short time. Bone marrow is found in the medullary canals of long bones and in the cavities of cancellous bones.Two types of bone marrow have been described based on their appearance on gross examination:
1-red, or hematogenous, bone marrow, whose color is produced by the presence of blood and blood-forming cells; and
2-yellow bone marrow, whose color is produced by the presence of a great number of adipose cells.
In newborns, all bone marrow is red and is therefore active in the production of blood cells. As the child grows, most of the bone marrow changes gradually into the yellow variety. Under certain conditions, such as severe bleeding or hypoxia, yellow bone marrow is replaced by red bone marrow.
Red Bone Marrow
Red bone marrow (Figure 1) is composed of a stroma (from Greek, meaning bed), hematopoietic cords, and sinusoidal capillaries.
The stroma is a three-dimensional meshwork of reticular cells and a delicate web of reticular fibers containing hematopoietic cells and macrophages. The stroma of bone marrow contains collagen types I and III, fibronectin, laminin, and proteoglycans. Laminin, fibronectin, and another cell-binding substance, hemonectin, interact with cell receptors to bind cells to the stroma.
The sinusoids are formed by a discontinuous layer of endothelial cells.
Section of active bone marrow (red bone marrow) showing some of its components. Five blood sinusoid capillaries containing many erythrocytes are indicated by arrowheads. Note the thinness of the blood capillary wall. Giemsa stain. Medium magnification.
Maturation of Erythrocytes
A mature cell is one that has differentiated to the stage at which it has the capability of carrying out all its specific functions. The basic process in maturation is the synthesis of hemoglobin and the formation of an enucleated, biconcave, small corpuscle, the erythrocyte. During maturation of the erythrocyte, several major changes take place.
These Changes are :
1- Cell volume decreases, and
2- The nucleoli diminish in size until they become invisible in the light microscope.
3- The nuclear diameter decreases, and
4- The chromatin becomes increasingly more dense until the nucleus presents a pyknotic appearance and is finally extruded from the cell.
5- There is a gradual decrease in the number of polyribosomes (basophilia decreases),
6- A simultaneous increase in the amount of hemoglobin (an acidophilic protein) within the cytoplasm.
7- Mitochondria and other organelles gradually disappear .
There are three to five intervening cell divisions between the proerythroblast and the mature erythrocyte. The development of an erythrocyte from the first recognizable cell of the series to the release of reticulocytes into the blood takes approximately 7 days.
The hormone erythropoietin and substances such as iron, folic acid, and cyanocobalamin (vitamin B12) are essential for the production of erythrocytes. Erythropoietin is a glycoprotein produced mainly in the kidneys that stimulates the production of mRNA for globin, the protein component of the hemoglobin molecule.
Differentiation of erythrocytes
The differentiation and maturation of erythrocytes involve the formation (in order) of proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, orthochromatophilic erythroblasts (normoblasts), reticulocytes, and erythrocytes .
1- Proerythroblastis the first recognizable cell in the erythroid series. It is a large cell with loose, lacy chromatin and clearly visible nucleoli; its cytoplasm is basophilic.
2- The next stage is represented by the basophilic erythroblast, with a strongly basophilic cytoplasm and a condensed nucleus that has no visible nucleolus. The basophilia of these two cell types is caused by the large number of polyribosomes involved in the synthesis of hemoglobin.
3- During the next stage, polyribosomes decrease, and areas of the cytoplasm begin to be filled with hemoglobin. At this stage, staining causes several colors to appear in the cell—the polychromatophilicerythroblast.
4- In the next stage, the nucleus continues to condense and no cytoplasmic basophilia is evident, resulting in a uniformly acidophilic cytoplasm—the orthochromatophilicerythroblast.
5- At a given moment, this cell puts forth a series of cytoplasmic protrusions and expels its nucleus, encased in a thin layer of cytoplasm. The expelled nucleus is engulfed by macrophages. The remaining cell still has a small number of polyribosomes that, when treated with the dye brilliant cresyl blue, aggregate to form a stained network. This cell is the reticulocyte,
6- Reticulocyte which soon loses its polyribosomes and becomes a mature erythrocyte.
Buffer systems and how they work
Enzyme Carbonic anhydrase (CA) made acid/base equilibrium H2O/CA/CO2/NaHCO3
There are several important buffer systems, that act in the human organism and allow pH of the organism to be constant iarrow interval allowed changes (pH = 7.36) despite the fact, that organism produces great amount of acidic products – the amount of acidic products is equivalent to
HHb H+ + Hb– ( 0)
and salt form NaHb Na+ + Hb– ( = 1)
and remember, that the salt is a strong electrolyte and the presence of Hb ions due to salt shifts the dissociation equilibrium of the acid to the left, we can explain, why pH is not changed, when strong acid or base is added to buffer system. If acid is added, the H3O+ ions of the strong acid react with the basic component of the buffer – Hb– ion: H3O++ Hb– HHb + H2O
Thus, the strong acid (H3O+) is transformed into a weak one (HHb) and, as the dissociation degree of the weak acid decreases with the growth of concentration, H+ concentration and, hence, pH will remain constant. When a base is added to buffer system, it will react with the buffer acid – HHb:
In such a way, the strongest possible base in water solution – the OH– – ion is transformed into a buffer Brønsted base – salt Hb– ion. HHb is used for this, but, as grows the HHb concentration, with the decrease H+ concentration pH will remain the same. (This “chemical” mechanism of buffer action is absolutely similar to the mechanism of any other buffer system that is composed from a weak acid and its salt, therefore it will not be repeated for all the following biological buffer systems.) Second buffer system of the human blood is oxyhemoglobin buffer system HHbO2/NaHbO2. Oxyhemoglobin is a product of hemoglobin reaction with oxygen, or, in other words, it is a molecule of hemoglobin, that has bound a molecule of oxygen.
3) a very important buffer system of the human organism is the hydro carbonate buffer system, where carbonic
anhydrase(CA) keeps CO2 as acid and base salt is the bicarbonate ion HCO3– respectively H2O/CA/CO2/NaHCO3 which equilibrium constant pK=7.0512 value very close to blood buffer systems made pH=7.36.
4) Next buffer system, that is present in blood, is the protein buffer system. This one has to be explained a little more, as it differs from the usual buffer systems that are composed from an acid and a salt. A protein is a long chain of amino acid remainders, but this long chain still has free carboxylic groups and free amino groups. For this reason here and further for general explanations about proteins we will use for it a following notation:
showing both free functional groups of protein molecule in such a way.
As the carboxylic group has acidic properties and the amino group – basic properties, no other component of buffer system is required – a protein molecule itself can stand both addition of an acid or a base. If an acid is added to solution, containing protein, the H3O+ ions will react with the amino group:
and the strong acid will be transformed into a weak one.
If a strong base is added to protein-containing solution, OH– ions react with the carboxylic groups of protein:
5) The next important biological buffer system is the phosphate buffer system
NaH2PO4 / Na2HPO4.
6) Besides the inorganic phosphate buffer system, a buffer system of the organic esters of phosphoric acid also exists:
(If there are any difficulties to understand the structure of last two compounds, remember, that phosphoric acid can be shown in structure as
In the ester of phosphoric acid one of the hydrogen atoms is replaced by an organic radical. Practically the buffer system consists of a mono substituted and bi substituted salts of the ester). Not all of these 6 buffer systems act in the same place.
In erythrocytes the main buffer systems are both hemoglobin-based buffer systems and hydrogen carbonate buffer system.
In blood plasma – hydrogen carbonate, protein and phosphate buffer systems.
In sweat, urine and digestive apparatus, the phosphate system is the main one.
Besides the normal “chemical” mechanisms of buffer action in maintaining constant pH=7.36, hemoglobin, oxyhemoglobin and by carbonic anhydrase CA driven hydrogen carbonate buffer systems have a joint physiological mechanism of action, which carries out the exchange of breathed in O2 and breathed out CO2 between air in lungs and tissues and environment of human body.
PHYSIOLOGICAL MECHANISM breathed in O2 and breathed out CO2 ACTION OF HEMOGLOBIN, OXYHEMOGLOBIN AND HYDROCARBONATE BUFFER SYSTEMS
Before any discussion of the mechanism, we have to know the sequence of strengths of the three acids, involved in the three buffer systems. The strongest one of these three acids is oxyhemoglobin, next one is carbonic anhydrase CA made acid with value pK=7.0512 and the weakest one is hemoglobin: KHHBO2>KH2O/CA/CO2>KHHb
(The sequence of acid strength will be necessary for further explanation).
Venous blood, which flows to lungs, contains two components of these buffer systems –
NaHCO3 and HHb (NaHCO3 is a transport form of CO2).
As soon as a portion of venous blood reaches lungs, the following processes occur:
Processes in lungs on cell wall membrane aquaporins penetrating water and oxygen
1) HHb + O2 HHbO2
2) As the oxyhemoglobin acid, which is formed in this process, is stronger than H2O/CA/CO2,
it starts to react with NaHCO3:HHbO2 + NaHCO3 Na++HbO2– + H+ + HCO3–
Carbonic anhydrase turn back carbonic dioxide to bicarbonate anion H++HCO3–/CA/H2O+CO2:
Carbonic acid H++HCO3–H2CO3on lung epithelial cell surface with absence carbonic anhydrase made equilibrium is unstable and decomposes outside cell: membraneH+ + HCO3– H2CO3 H2O + CO2
This transport H+ HCO3– is catalyzed by a special enzymes bicarbonate HCO3– and proton H+ channels (pumps), which are a transport enzymes. The epithelial cell surface of lungs has the specific building: super thin 0.6 nm water layer on surface 9·105 nm2 S=950 nm x 950 nm within small volume 0.5·106 nm3 creates acidity increase up to pH=5.5 if one proton crosses the membrane channel reaching the surface and that cause fast evolving CO2 gas breathed out, because otherwise blood flows away from lungs, while carbonic acid is not completed and decomposed outside cell. CO2, liberated on last step reaction on epithelial cell surface breathed out, but oxygen O2, which adsorbed on hemoglobin transport form NaHbO2 transported to tissues in human body. It is necessary to increase the removing rate of bicarbonate and hydrogen ions out of cells.
For the reasons, discussed above, arterial blood contains NaHbO2, [CO2]=0.0076M and [HCO–3]=0.0154 M total sum which releases 56.23 mL of gaseous CO2 on 100 mL of blood sample.
As soon as the arterial blood reaches tissues, the following reactions occur. Processes in tissues
1) NaHbO2 loses oxygen if blood oxygen concentration [O2] =6·10–
2) CO2, which is a product of metabolism comes from tissues and is dissolved in blood. In blood it reacts with water, forming carbonic anhydrase made equilibrium: CO2 + 2H2O CA H3O++HCO3–
Carbonic anhydrase equilibrium constant pK=7.0512 shifts reaction towards bicarbonate anion to prevent of carbonic dioxide accumulation, according Le Chatelier’s due to high water H2O concentration
3) As carbonic anhydrase made equilibrium acid is a stronger acid, than HHb, bicarbonate and hydrogen ion reacts with NaHb:
H++HCO3– + NaHb NaHCO3 + HHb
In this way, we have got back the content of venous blood – we have followed one full cycle of the process.
Let us consider now, why this sequence of acid strengths (given in the beginning) is necessary.
First, if it happened, that H2O/CA/CO2, was a stronger acid, than HHb, gas CO2 would stay in its water soluble transport form NaHCO3– and could not be liberated up to in lungs via bicarbonate HCO3– and proton H+ channels out of cells to epithelial lung cell surface having absent carbonic anhydrase.
Second, if HHb was a weaker acid than H2O/CA/CO2, HHb not react with NaHCO3 and is transported to lungs in the form of bicarbonate. Opposite is dangerous, because accumulation and formation of CO2 bubbles could occur in blood vessels, thus interfering the blood circulation.
VIII. pH OF BLOOD
As it was mentioned before, three main buffer systems act in blood: HHb/NaHb
HHbO2/NaHbO2
H2O/CA/CO2/NaHCO3
When these buffer systems struggle with acidic products of metabolism, more and more of the acid forms of the buffer systems are produced. For this reason, the acid forms have to be transported out of organism.
It is easy to imagine, that hemoglobin cannot be evolved out of organism, therefore there is only one buffer system, suitable for regulation of acid form’s presence by breathing out CO2, that decrease metabolic acid production caused problems for organism.
Carbonic anhydrase equilibrium constant pK=7.0512 decreases concentration acid form CO2 into water H2O (avoid carbonic acid H2CO3 formation) and hydrogen carbonate HCO–3 + hydrogen ion H+ are included into equation for blood pH:
pH = 7.0512 +log = 7.36; =
the ratio [NaHCO3]/[CO2] being approximately 2/1. (usually in medical literature CO2 concentration is given, but as 1 mole CO2 creates 1 mole H2O/CA/CO2, it is the same).
There is a chain of equilibria, which have to be shifted from carbonic anhydrase made equilibrium to transport CO2 out as gas: CO2(gas) CO2(dissolved) CO2 + 2H2O CA H3O++HCO3–
The gaseous CO2, which is contained in the alveolar air, is in equilibrium with CO2, dissolved in blood. The dissolved into water H2O carbonic dioxide CO2 occurring in cell converted with carbonic anhydrase CA to
H+ + HCO3–, that H2O carbonic dioxide CO2, finally, is in direct equilibrium with its ions H+and HCO–3.
A– 0% 50% 100% salt – buffer system base
HA 100% 50% 0% weak acid buffer component
As soon as H+ concentration grows for some reason, all the chain of equilibriums is shifted to left and CO2 transported out by breathing. If H+ concentration decreases, all the equilibriums are shifted to the right and the extra amount of HCO–3 through kidneys passes into urine and is transported out.
The numerical value
The alkaline reserve 2.036/1=[HCO–3]/[CO2] of the organism can be controlled by adding H2SO4 to a sample of blood (H2SO4 reacts with NaHCO–3 and the CO2, included in salt, is liberated). If 56.23 mL of gaseous CO2 are liberated from 100 mL of blood, the alkaline reserve is normal and total alkaline reserve amount concentration 0.023M = [HCO–3]+[CO2] is normal as [HCO–3] =
Controlled instructions the alkaline reserve of the organism by adding H2SO4 to a sample of blood
(H2SO4 reacts with NaHCO–3 and the CO2, included in salt, is liberated).
If 50–60 mL of gaseous CO2 is liberated from 100 mL of blood, the alkaline reserve is normal.
Two types of diseases occur, if the acid-base balance is distorted in the organism alkalosis and acidosis.
1) Respiratory alkalosis occurs, if lungs are hyperventilated, for example, during anesthesia. If CO2 concentration decreases due to hyperventilation, the blood vessels are broadened and their tonus is lowered as a result of it, therefore O2 supply to brain is shortened.
For this reason it is necessary to use mixtures of O2 and CO2 during anesthesia instead of pure oxygen. If respiratory alkalosis occurs for other reasons than hyperventilation of lungs, the ratio 2/1 of the buffer components can be re-established in a longer period of breathing normal, CO2-containing air 350 ppm.
2) Respiratory acidosis occurs in the cases, when the concentration of CO2 in the air is increased. The result of this is that the action of breathing muscles becomes more difficult. Again, this can be canceled, if the patient starts breathing normal air. Hoverer, if increased CO2 content in the air lasts long, a metabolic acidosis can occur. In the case of metabolic acidosis the ability of hemoglobin to bound oxygen is lowered.
For this reason only the concentrations of carbonic dioxide CO2 into water H2O (avoid carbonic acid H2CO3 formation) and hydrogen carbonate HCO–3 + hydrogen ion H+ are included into equation for blood pH:
pH = 7.0512 +log = 7.36 ; =
the ratio [NaHCO3]/[CO2] being approximately 2/1. (usually in medical literature CO2 concentration is given instead of H2O/CA/CO2, but as 1 mole CO2 creates 1 mole H2O/CA/CO2, it is the same).
There is a chain of equilibria, which have to be shifted for transporting CO2 out:
CO2(gas) CO2(dissolved) CO2 + H2O CA H+ + HCO3–
The gaseous CO2, which is contained in the alveolar air, is in equilibrium with CO2, dissolved in blood. The dissolved into water H2O carbonic dioxide CO2 occurring in cell converted with carbonic anhydrase CA to
H+ + HCO3–, that H2O carbonic dioxide CO2, finally, is in direct equilibrium with its ions H+and HCO3–.
Age features of physical and
chemical properties of the blood
Newborns and babies of the first year have different features of the blood than adults. So, newborns have higher density and stickiness of the blood, which is determined by higher erythrocytes concentration. Till the end of the first month of life, these features decrease and approach to the one, adults have, or they become lower.
Placental blood circulation and labor complicate interchange of gases. That is why children have acidosis before birth (pH = 7,13 – 7,23). During the first hours (or days) after birth, acidosis gradually disappears.
The concentration of plasma proteins iewborn organism is lower (50 – 56 g/l). It will reach the level of adult in age of 3 – 4 years. The high concentration of γ-globulins is specific for the newborn, which the newborn gets from the mother. Till the end of third month its content decreases, but in future, with the help of its own antibodies formation, it will gradually increase. The concentration of α and γ-globulins reaches the adult level till the end of the first year of life.
With age, most of the physical and chemical properties of the blood (pH, osmotic pressure, sodium and potassium concentration, viscosity), stay on the same level. Other features can change. So, ECR increases, osmotic resistance of erythrocytes, hematocrit, ablolute and relative albumins concentration decrease.
APPENDIX
I. Blood plasma content values
Inorganic part:
Fe (iron) 8,53 – 28,06 mkmol/l
K (potassium) 3,8 – 5,2 mmol/l
Na (sodium) 138-217 mkmol/l
Ca (calcium) 0,75 – 2,5 mkmol/l
Mg (magnesium) 0,78 – 0,91 mkmol/l
P (phosphorus) 0,646 – 1,292 mkmol/l
Chlorides of blood 97 – 108 mkmol/l
Filtrate nitrogen (not-protein) 14,28 – 25 mkmol/l
Urea 3,33 – 8,32 mmol/l
Creatinine 53 – 106,1 mkmol/l
Creatine Men 15,25 – 45,75 mkmol/l
Women 45,75 – 76,25 mkmol/l
Uric acid Men 0,12 – 0,38 mkmol/l
Women 0,12 – 0,46 mkmol/l
Organic part:
Total protein 65 – 85 g/l
Albumins 35 – 50 g/l
(52 – 65%)
Lactatedehydrogenase (LDH) < 7 mmol (hour/l)
Aldolase 0,2 – 1,2 mmol (hour/l)
α-amilase (diastase of blood) 12 – 32 g/l (hour/l)
Aspartateaminotransferase (AST) 0,1 – 0,45 mmol (hour/l)
Alaninaminotransferase (ALT) 0,1 – 0,68 mmol (hour/l)
Cholinesterase 160 – 340 mol (hour/l)
Basic phosphatase 0,5 – 1,3 mmol (hour/l)
Creatinkinase 0,152–0,305mmol (hour/l)
Creatinphosphokinase (KPK) to 1,2 mmol
Lipase 0,4 – 30 mmol (hour/l)
Globulins 3 – 35 g/l (35 – 48%)
Total bilirubin 8,5 – 20,5 mkmol/l
free bilirubin
(indirect, not conjugated) 1,7 – 17,11 mkmol/l
conjugated bilirubin (direct) 0,86 – 5,1 mkmol/l
Lipids (total amount) 5 – 7 g/l
Triglicerids 0,59 – 1,77 mmol/l
Total cholesterol 2,97 – 8,79 mmol/l
Lipoproteins of very low density 1,5 – 2,0 g/l
(0,63 -0,69 mmol/l)
low density 4,5 g/l
(3,06 – 3,14 mmol/l)
high density 1,25 – 6,5 g/l
(1,13 – 1,15 mmol/l)
Chylomicrons 0 – 0,5 g/l
(0 – 0,1 mmol/l)
Glucose of the blood 3,3 – 5,5 mmol/l
Glycolized hemoglobin 4 – 7%
II. Normal Values for Erythrocyte and Leukocyte
Measurements
Hemoglobin 13–18 g/dL (males);
12–16 g/dL (females)
Hematocrit 42–52% (males);
37–48% (females)
Erythrocyte count (male) 4.5–6.0 × 106/mm3
(females) 4.0–5.5 ×106/mm3
Leukocyte count 5 × 103–10 × 103/mm3
Differential Leukocyte Count
Neutrophils 55–75%
Eosinophils 2–4%
Basophils 0.5–1%
Lymphocytes 20–40%
Monocytes 3–8%
Short review:
The Blood
Fluids of the Body
Cells of the body utilize 2 fluids:
Blood
Composed of plasma and a variety of cells
Transports nutrients and wastes
Interstitial fluid
Bathes the cells of the body
Nutrients and oxygen diffuse from the blood into the interstitial fluid & then into the cells
Wastes move in the reverse direction
Functions of Blood
Transportation
O2, CO2, metabolic wastes, nutrients, heat & hormones
Regulation
helps regulate pH through buffers systems (discussed in later chapters)
Carbonic-Acid-Bicarbonate Buffer System
Phosphate buffer system
Protein buffer system
helps regulate body temperature
H2O in plasma has high specific heat capacity, buffering large fluctuations in temp
Vessels direct warm blood to where it’s needed, or to the skin for heat dissipation
Protection from disease & loss of blood
Physical Characteristics of Blood
Thicker (more viscous) than water, and flows more slowly than water
Temperature of
pH 7.4 (7.35 – 7.45)
If pH 7 is neutral, blood at 7.4 is slightly alkaline
Average Blood volume:
Females: 4 –
Males: 5 –
Hormonal negative feedback systems maintain constant blood volume and pressure
Components of Blood
55% plasma
45% cells
99% RBCs
< 1% WBCs and platelets
Hematocrit (Hct) & Hemoglobin (Hb)
Hematocrit (Hct) – percentage of blood volume occupied by RBCs
volume of red blood cells ÷ total blood volume
Normal Hematocrit range:
adult female: 38 – 46% (average of 42%)
adult male: 40 – 50% (average of 45%)
Hemoglobin (Hb) – the protein responsible for transporting oxygen in the blood
Normal Hemoglobin range:
adult females: 12 – 16 g/100mL of blood
adult males: 13.5 – 18 g/100mL of blood
Anemia – not enough RBCs, hemoglobin
Polycythemia – too many RBCs (over 50%)
Blood Plasma
Over 90% water
7% plasma proteins
created in liver
confined to bloodstream
albumin
Blood osmotic pressure
Transporter substances
globulins
Immunoglobulins (antibodies)
Defense against foreign proteins
fibrinogen
Clotting protein precursor
2% other substances
electrolytes, nutrients, hormones, gases, waste products
Formed Elements of Blood
Red blood cells (erythrocytes)
Platelets (thrombocytes)
White blood cells (leukocytes)
granular leukocytes
Neutrophils
Eosinophils
Basophils
agranular leukocytes
lymphocytes (T cells, B cells, and natural killer cells)
monocytes
Formed Elements of Blood
Normal RBC count: ~ 5 million/drop
Males: 5.4 million/drop
Female: 4.8 million/drop
Platelet count: 150,000-400,000/drop
WBC count: 5,000 – 10,000/drop
Ratio:
RBC : Platelet : WBC
700 : 40 : 1
Hematopoiesis: Formation of Blood Cells
Most blood cell types need to be continually replaced
Blood cells die within hours, days, or weeks
Hematopoiesis (or hemopoiesis) – the process of blood cell formation
In adults
Occurs only in red marrow of flat bones (pelvis, sternum, ribs, vertebrae, & skull, and in ends of long bones)
Hematopoiesis of All Blood Cells
All blood cells develop from the same uncommitted stem cells in bone marrow
Red Blood Cells or Erythrocytes
Contain oxygen-carrying protein hemoglobin that gives blood its red color
1/3 of cell’s weight is hemoglobin
Biconcave disk
Increased surface area:volume ratio
Flexiblity for narrow passages
No nucleus or other organelles
No mitochondrial ATP formation
New RBCs enter circulation at 2-3 million/second
Hemoglobin
Hemoglobin Molecule:
>> 4 globular protein subunits
>> each containing 1 heme group (red pigment)
>> each containing 1 iron ion (Fe+2)
>> each capable of binding (reversibly) to 1 oxygen (O2) molecule
1 RBC = ~ 280 million Hemoglobins
1 Hemoglobin = 4 Heme Groups
1 Heme Group = 1 Iron atom = 1 O2 molecule
Therefore, 1 RBC contains ≈ 1.12 x 109 O2 molecules
Function of Hemoglobin
Each hemoglobin molecule can carry 4 O2 or CO2 molecules
Hemoglobin also acts as a buffer and balances pH of blood
Hemoglobin transports 23% of total CO2 waste from tissue cells to lungs for release
combines with amino acids in globin portion of Hb
Forms of Hb:
Oxyhemoglobin: hemoglobin + O2
Deoxyhemoglobin: hemoglobin – O2
Carbaminohemoglobin: hemoglobin + CO2
Hemoglobin Affinity
CO2 vs O2
Deoxyhemoglobin’s affinity for carbon dioxide (CO2) is greater than its affinity for oxygen (O2)
Carbon dioxide (CO2) can lower O2-Hb affinity through changes in its partial pressure (pCO2) or pH (carbonic acid reaction)
CO vs O2 (Carbon Monoxide Poisoning):
Hemoglobin’s affinity for carbon monoxide (CO) is 250 times greater than its affinity for oxygen (O2)
CO is colorless, odorless, flammable, and highly toxic
CO binds irreversibly to the Fe2+ in hemoglobin. Treatment requires oxygen therapy, or hyperbaric oxygen therapy, depending on severity of poisoning
The drop in Hb O2 saturation goes unnoticed for a while because chemoreceptors rely primarily on [CO2] for the “urge to breathe”
Erythropoiesis: Production of RBCs
Multipotent stem cell differentiates into Proerythroblast
Proerythroblast begins producing hemoglobin, becoming erythroblast
Erythroblast ejects nucleus, becoming a reticulocyte
Reticulocyte escapes from bone marrow into the blood.
In 1-2 days, reticulocyte ejects remaining organelles, becoming Erythrocyte
Factors required for Erythropoiesis:
Erythropoietin (EPO) from kidneys
Vitamin B12 (cobalamin)
Iron (Fe)
Negative Feedback Control of Erythropoiesis
Hypoxia – inadequate oxygen supply to tissues
generalized hypoxia – systemic oxygen deprivation
tissue hypoxia – local oxygen deprivation
Common Causes:
High altitudes
decreased atmospheric pressure
Anemia
RBC or hemoglobin production < RBC destruction
Kidney response to hypoxia:
Release Erythropoietin (EPO)
Speeds up cell division of erythroblasts, and maturation and release of erythrocytes
Negative Feedback Control of Erythropoiesis
Negative Feedback Control of Erythropoiesis
RBC Life Cycle
