Osmotic Fragility of RBCs PDF

Osmotic fragility of the RBCs Dr. Enas Sabah MSc. Medical Physiology Background • When cells are placed in hypotonic solutions water enters the cell due to osmosis and causes the cells to swell. When cells are distended beyond a limit, the cell membrane is stretched and the contents of the cell l...

Osmotic fragility of the RBCs Dr. Enas Sabah MSc. Medical Physiology Background • When cells are placed in hypotonic solutions water enters the cell due to osmosis and causes the cells to swell. When cells are distended beyond a limit, the cell membrane is stretched and the contents of the cell leak out. This effect can be easily detected when red cells are studied because haemoglobin leaks out and forms a clear pink solution (haemolysis). Shape and characteristics of Red Blood Cells. • Normal red blood cells are biconcave discs, but the shapes of RBCs can change remarkably as the cells squeeze through capillaries. • because the normal cell has a great excess of cell membrane for the quantity of material inside, deformation does not stretch the membrane greatly and, consequently, does not rupture the cell. Osmotic fragility •It is defined as the ease with which the cells are broken down in hypotonic solutions. It is expressed in terms of concentration of saline solutions in which cells are hemolysed. •It is affected by various factors, like membrane composition and integrity as well as surface-area to volume ratio. •The osmotic fragility test is often performed to aid with diagnosis of diseases associated with RBC membrane abnormalities. Osmotic Pressure and Tonicity • Osmosis is the movement of water molecules through a semipermeable membrane from a region of high water concentration to low water concentration is called osmosis that tends to equalize the concentrations of solute on the two sides of the membrane. • Osmotic pressure its the pressure that would be required to stop water from diffusing through a semipermeable by osmosis. Tonicity is the measure of this pressure. Procedure: 1. Take 8 small test tubes and keep them in a rack. 2. Put 5 ml of different saline concentrations (0.9%, 0.7%, 0.6%, 0.55%, 0.45%, 0.35%, 0.3%, and 0.2%) to their corresponding labeled test tubes. 3. Draw 1 ml of blood and then put exactly equal drop of blood to inside each test tube. 4. Shake each test tube gently to mix the blood with the solution. 5. Spin the test tubes inside the centrifuge at around 10000 RPM for 5 minutes. 6. Find the value at which the hemolysis is just starting and the value at which the hemolysis is complete. Observation and Results •Normally hemolysis begins between 0.55-0.45% saline solutions and becomes complete at around 0.35%. • The indicator of partial hemolysis is turning of colour of the solution to red in addition to deposition of part of the cells in the bottom of the test tubes. •Complete hemolysis is indicated by red-colored solution without any deposition. Observation and Results • Note the test tubes in which no hemolysis has occurred, the RBCs will settle down and form a red dot (mass) at the bottom of the tube, leaving the saline above clear. • If there is some hemolysis, the color of saline will change to red because of Hb leaks out the cell, with the unruptured cells forming a red dot at the bottom. The color of the saline will be seen to be increasingly deeper with decreasing tonicity of saline. The test tubes in which there is complete hemolysis, the saline will be equally deep red with no red cells at the bottom of these tubes. Medical applications •Fragility of erythrocytes increases in: 1. hereditary spherocytosis 2. glucose-6-phosphate dehydrogenase (G6PD) deficiency. 3. Autoimmune hemolytic anemia. (Autoimmune antibodies damage the structural proteins). •Note: when we say that fragility of RBCs increase that’s mean break down of RBCs increase. Medical applications • Osmotic fragility decreased in: • Thalassemia. • Iron deficiency anemia, why? • Sickle cell anaemia Hereditary spherocytosis • The disorder is caused by mutations in genes relating to membrane proteins. • The abnormal erythrocytes are spherical rather than the normal biconcave disk shaped, this abnormal shape interfere with the cell's ability to be flexible to travel from the arteries to the smaller capillaries. • This difference in shape also makes the red blood cells more prone to rupture. Glucose-6-phosphate dehydrogenase (G6PD) deficiency • G6PD deficiency is a genetic abnormality that results in an inadequate amount of glucose-6-phosphate dehydrogenase (G6PD) in the blood and occurs almost in males. • The inherited disorder caused by a genetic defect in the red blood cell (RBC) enzyme G6PD, protects RBCs from oxidative injury. • Some people may develop symptoms when they’re exposed to the medication, food, or infection that triggers the early destruction of red blood cells. Once the cause is treated or removed, symptoms of G6PD deficiency usually relieve.