Questions for discussion.
1. Blood as a system. The hematocrit, also known as erythrocyte volume fraction (EVF)
2. Plasma composition. Physico-chemical constants of blood: osmotic pressure, oncotic pressure, blood pH, viscosity, specific gravity, erythrocyte sedimentation rate.
3. Erythrocytes –structure, function. RBCs – functions: transport gases, maintenance of acid-base balance. Osmotic resistance of RBCs. Determination method
4. Hemoglobin, structure, functions. Compounds of hemoglobin, it types, color index. Determination method of hemoglobin concentration
Books recommended
1. Ganong W.F. Review of Medical Physiology. 20thed; McGraw-Hill Companies, Inc., 2001. – P.
2. Guyton A.C., Hall J.E. Textbook of Medical Physiology, 12thed; WB Saunders, 2008.
Practical work 1. Determination of osmotic resistance of erythrocytes (demonstrational work).
For this work you need: donor blood, a tripod with 8 tubes which each of them are filled with 2 ml NaCl solution of different concentration (0,8; 0,7; 0,6; 0,5; 0,4; 0,3; 0,2; 0,1%); capillary (from Sally’s hemometer with 0,02 ml segmentation), rubber bag, rubber gloves, wadding.
Technique. Place 0,02 ml blood on each tube by using capillary, mix and leave for 30 min.
Results. Draw the results, point concentration of solution of partly and full (as known as «laky blood» - pellucid and glistening) hemolysis of erythrocytes.
Conclusions. Compare your results with minimum and maximum values of normal osmotic resistance of red blood cells.
Grid in the camera Goryaeva
\ 
Practical work 2. Counting red blood cells in Goryaev'schamber.
For this work you need: donor blood, Goryaev's chamber, a cover glass, a test tube, a capillary (from the hemometerSali), a microscope with two lenses (x8, x40), a rubber pear, a 5-ml pipette, 3% NaCl solution, rubber gloves, cotton wool.
Technique. Attach the cover glass to the Goryaev’schamberto create a 0.1 mm high camera above the mesh. In a test tube, pour 4 ml of a 3% NaCl solution. In the capillary (from the hemometerSali), collect donor blood untill the mark (0.02 ml), transfer it to a test tube, mix (the blood was diluted 200 times), place the diluted blood into the Goryaev’s chamber by using a glass rod. In a small increase in the microscope, find the grid in the chamber, find the left upper large square divided by small, place it into the center of the field of view and put a large increase. Count the erythrocytes in 5 large squares which are divided into 16 small ones. Choose large squares diagonally for counting the red blood cells. In each large square, count the red blood cells in order of its small squares, using Egorov's rule (squares include red blood cells located on its area and on its left and upper sides).
The formula for counting the amount of erythrocytes:
Erythrocytes = A * 200 * 4000 * 106 /80,
Where A - the total number of erythrocytes in 5 large squares, 80 - number of small squares, 200 - dilution, 4000 - is the number of times the volume of liquid over a small square of smaller volume 1 mm3 (square side - 1/20 mm and 1/10 mm chamber height), 106 - forconverting values in a volume of 1 liter.
Results. Draw a grid with large and small squares, draw 5 large squares divided into small ones, depict red blood cells found in each small square, add up all the values, write down the number A, calculate the number of red blood cells according to the formula.
Conclusions. Compare the result with the norm; determinehow is called the deviation from the norm to the larger and smaller sides?
Practical work 3. Determination of the amount of hemoglobin by Sally’s method.
For this work you need: donor blood, Sally’shemometer, capillary for 0.02 ml of blood, pipette, 0.1% HCl solution, cotton wool, distilled water, glass rod, rubber gloves.
Technique. In the middle tube of the Sally’s device, pour 0.1% HCl solution until the lower mark, draw blood from the bottle (0.02 ml) and draw it into the acid solution without removing the capillary, rinse it with acid, mix the contents of the tube, place it in a tripod for 5-10 minutes. This time is necessary for the complete conversion of hemoglobin into a dark brown colored acid Hematin. Then add the distilled water dropwise to the contents of the tube, each time mixing with the solution by a glass rod. Bring the color of the test solution to the color of the standard.
Results. Read the number on the middle tube of the Sally’s instrument, which corresponds to the level of the obtained solution, record the result. Point the amount of hemoglobin in g/l because the Sally’s device gives it in g per 100 ml of blood.
Conclusions. Compare the result with the normal amount of hemoglobin.
Practical work 4. Determination of the amount of hemoglobin by photometric method byusing ahemoglobinometerMiniGEM.
For this work you need: photometric hemoglobinometer, distilled water, ammonia solution 0,04%, scarifier, capillary for Sally’s hemometer, alcohol, iodine, cotton wool, 2 test tubes.
Technique. 1) Optical zero setting. A clean cuvette is filledby distilled water and make density measurement in the instrument. If the scale of the instrument shows values from -1 to +1, further research is carried out. 2) Study of hemoglobin. 2 ml of 0.04% ammonia solution are poured into two test tubes. Take the blood by using capillary for the Sally’shemometeruntilthe mark and pour into each tube, mix thoroughly. The mixture is poured into an optical cuvette from one tube (the second is for reliability), the cuvette is placed into the cell of the device. After the sound signal appears a number corresponding to the concentration of hemoglobin in 1 liter of blood.
Results. Write the result in a notebook.
Conclusions. Compare the result with the normal amount of hemoglobin.
Practical work 5. Determination of the saturation of erythrocytes by hemoglobin.
5.1. Calculation of the hemoglobin (Hb) content in one erythrocyte (HCE).
Technique. Calculate according to the formula:
HCE = The amount of Hb in 1 liter of blood / The number of red blood cells in liters of blood.
Results. Expressthe result in pg, 1 g - 1012pg.
Conclusions. Compare the result with the normal value.
5.2.Calculation of the color index of blood (CI).
Technique. Calculate according to the formula:
CI = (Experimental amount of Hb (in g/l) / 167 (g/l)): (Experimental amount of erythrocytes (in l) / 5 * 1012(in l)).
Results. Record the result of the calculation.
Conclusions. Compare the result with the normal value of the amount of hemoglobin.
It fluctuates between 0.8 - 1 unit.
If less than 0.8 - Hypochromia,
If more than 1 -Hyperchromia.
Terms (Blood)
1.H(а)ematocrit (гематокрит) - erythrocyte volume fraction (EVF), is the volume percentage (%) of red blood cells in the blood.
2. Osmosis (осмос) -movement of water from lower concentration to higher concentration solution across semi-permeability membrane.
3. Оsmotic pressure (осмотическое давление) - is defined as the minimum amount pressure needed to prevent osmosis.
4. Oncotic pressure (онкотическое давление), or colloid osmotic pressure - is a form of osmotic pressure exerted by blood plasma proteins.
5. Osmotic resistance of RBCs (осмотическая резистентность эритроцитов) - concentration at which complete hemolysis of erythrocytes occurs
6. Blood viscosity (вязкость крови) can be described as the thickness and stickiness of blood.
7. Homeostasis (гомеостаз) - the ability or tendency of organism or cell to maintain internal equilibrium by adjusting its physiological processes.
8. Protective (защита) - immunity and non-specific resistance; - blood coagulation
9. H(a)emopoiesis (гемопоез) - the formation of blood cellular components.
10. Serum (сыворотка) - plasma without fibrinogen
11. Isotonic solution (изотонический раствор) - it has osmotic pressure equal of blood plasma (NaCl 0.9 % and also glucose 5 %)
12. Hypotonic solution (гипотонический раствор) < (0.9 % NaCl)
13. Hypertonic solution (гипертонический раствор) > (0.9% NaCl)
14. pH of blood (рН крови) - the negative decimal logarithm of hydrogen concentration
15. Erythropoiesis (эритропоэз) - the process by which red blood cells (erythrocytes) are produced.
16. Erythrocytosis (эритроцитоз) - if the erythrocyte count is more than normal, such state is called
17. H(a)emolysis (гемолиз) - the rupturing of erythrocytes and the release of their contents (cytoplasm) into surrounding fluid (blood plasma).
18. Phagocytosis (фагоцитоз) -cellular ingestion of bacteria with enzymes proteases, peroxidases, cationic proteins
19. Immunity (иммунитет) - the capability to resist from pathogens, that tend to damage the tissues or organs, through biological defense.
20. Leucocytosis (лейкоцитоз) - increased amount of leucocytes in blood. (physiological: food intake, exercises, emotion, stress)
21. Anemia (анемия) - сondition of the blood in which the number of functional red blood cells or their hemoglobin content is below normal
22. Antibody (антитело) - а protein produced by plasma cells in response to a specific antigen; the antibody combines with that antigen to neutralize, inhibit, or destroy it. Also called an immunoglobulin
23. Antigen (антиген) - а substance that has the ability to provoke an immune, response and the ability to react with the antibodies or cells that
result from the immune response; contraction of antibody generator or Ig.
24. Hemostasis (гемостаз) - the stoppage of bleeding
Constants of blood.
1.pH of arterial blood – 7,40, venous blood – 7.36
2.Osmotic pressure - 7,6 atm.
3.Compound of plasma (состав плазмы): water – 92 %, protein – 7%, electrolytes, wastes, nutrients, vitamins, hormones -1%.
4. Plasma proteins include:
1) аlbumin - 65-85 g/l 2) globulin - 28 g/l 3) fibrinogen - 3 g/l
5. Glucose (глюкоза) - 3.3-5.5 mmol/L.
6. Specific gravity (плотность) of blood - 1.05 - 1.06 g/L
7. The hematocrit: 40-48% for men and 36-42% for women
8. Oncotic pressure -0.03-0,04 atm (or)20-25 mm Hg
9. Osmotic resistance of RBCs – 0,35 – 0,45 % NaCl
10. Blood viscosity – 5, plasma viscosity – 2,2
11.Leucocytes formula (лeйкоцитарная формула):
Granulocytes: Basophils - < 1%, Eosinophils -0,5-5%, Neutrophils – 47 -72 %
Agranulocytes: Monocytes - 3 -11%, Lymphocytes – 19 – 37%
12.The total blood volume makes up 6-8 percent of the body’s weight.
13.RBC сount: in male - 4.0-5.0 × 1012/L, in female - 3.5-4.5 × 1012/L
15.Normal range of haemoglobin: in men - 135-180 g/L, in women - 120-140 g/L
16. Normal count of WBC: 4-9 x 109/L.
17. Erythrocyte sedimentation rate for men 0 – 10 mm/h; for women 2 – 15 mm/h