In this test, the red cell fragility is tested by counting the cells in a hemocytometer, using 0.45% saline for diluting the blood in one pipette, and using Hayem’s fluid for diluting blood in a second pipette. Both pipettes are shaken for about 2 minutes and counts are made from both pipettes. The percent of red cells hemolysed in 0.45% saline is thus determined. Less than 20% of normal RBCs are hemolyzed by this method. In hereditary spherocytosis, the abnormal increase in fragility may cause hemolysis of more than 70% of red cells.
Precautions
1. Use the same dropper, after thorough rinsing each time, for measuring saline and distilled water.
2. The test tubes should not be shaken vigorously after adding blood, because this is likely to cause mechanical hemolysis.
3. The test tubes should be left undisturbed for one hour before making the observations.
Table 1.6: Preparation of saline solutions for testing the osmotic fragility of red cells
Test tube number 1 2 3 4 5 6 7 8 9 10 11 12
No. of drops of 1% NaCl 22 16 15 14 13 12 11 10 9 8 7 0
No. of drops of distilled water
3 9 10 11 12 13 14 15 16 17 18 25
Tonicity strength of NaCl (in%)
0.88 0.64 0.60 0.56 0.52 0.48 0.44 0.40 0.36 0.32 0.28 0 Note: Use the same dropper, after thorough rinsing each time, for measuring saline and distilled water. This will ensure that the volume of all drops is equal for all test tubes.
QUESTIONS
Q.1 What is meant by the terms fragility and hemolysis?
Fragility. This term refers to the susceptibility of red cells to being broken down by osmotic or mechanical stresses.
Hemolysis. This term refers to the breaking down (bursting) of red cells resulting in release of Hb into the surrounding fluid.
Q.2 Define osmosis and osmotic pressure. How much osmotic pressure is exerted by the blood and what is its importance?
Osmosis. It is the process of net movement of water from a weaker solution (of a solute and solvent) to a stronger solution through a selectively permeable membrane, that is permeable only to water but not to solute (salts, proteins, etc.).
Osmotic pressure. It is the pressure required to be applied to the stronger solution to prevent the movement of solute (water) from the weaker solution to the stronger solution. (It should be noted that the osmotic pressure does not produce the movement of water during osmosis).
Osmotic pressure of blood. The total osmotic pressure of blood (or plasma) due to all crystalloids and colloids is about 5000 mm Hg (6–7 atmospheres).
But since the crystalloids (mainly NaCl) are equally distributed across (on the two sides) the capillary walls, it is only the colloidal osmotic pressure exerted by plasma proteins (about 25 mm Hg) that takes part in tissue fluid exchanges. The colloid osmotic pressure opposes hydrostatic pressure (blood pressure) within the capillaries—about 32 mm Hg at their arterial ends and 12 mm Hg at the venous ends. As a result, filtration occurs at the arterial ends and reabsorption at the venous ends. Changes in these forces, called Starling forces, can cause edema (accumulation of fluid in the tissues).
Q.3 What will be the effect of vigorous shak-ing of the test tubes after adding blood to each of them?
Vigorous shaking, in an attempt to mix the contents of the test tubes, is likely to cause mechanical rupture of RBCs with release of Hb into the saline.
Q.4 How do red cells behave in hypotonic and hypertonic saline solutions? How do they resist hemolysis in hypotonic saline?
The red cell membrane is selectively permeable membrane which allows water to pass through easily while the movement of various solutes is restricted to varying degrees.
Red cells in hypertonic saline. In hypertonic solutions, the RBCs , like other body cells, shrink (crenate) due to movement of water out of the cells (exosmosis).
Red cells in hypotonic saline. In hypotonic saline, water moves into the red cells (endosmosis). They swell up and lose their biconcave shape, becoming smaller and thicker. When they swell and become completely spherical, further increase in volume is not possible without an increase in their surface area.
However, the surface area cannot increase because the cell membrane is “plastic” but not “elastic”, i.e., it can change shape but is not able to stretch. A completely round shape is reached when the red cell volume increases to about 150% of their original volume (say, from 90 µm3 to 140 µm3). It is clear that biconcave cells can resist greater hypotonicity as they can accommodate more and more water. Flat cells, on the other hand, can accommodate very small amounts of water before getting stretched and bursting.
Thus, osmotic fragility is an indicator of the shape of the cells. The more fragile the cells, the greater is their degree of spherocytosis. Also, fragility of red cells is greater in venous blood.
Comments
The red cell membrane has protein pumps and ion chan-nels. Its structural proteins, including spectrin, actin, tropomyosin, adducin, etc. are attached to the transmem-brane skeletal protein meshwork by the protein ankyrin.
(The Hb molecules are not present free within the cells but absorbed on to the protein meshwork). The structural proteins give the red cells the remarkable property of
“deformability” so that they can easily change their shape and squeeze through the 4-5 µm tissue capillaries and the still narrower and tighter meshwork and trabeculae of the spleen (in the spleen, which is an important blood filter which detains large and abnormal-shaped and rigid cells, part of the blood flows through the microvessels, while the rest ‘percolates’ through the phagocytes and lymphocytes of the splenic pulp before entering the sinusoids).
Q.5 Give the normal range of fragility of red cells.
See page 128.
Q.6 What will be the effect of waiting for 5–6 hours before observations are made on the test tubes?
If observations are made after, say, 5–6 hours, hemolysis is likely to occur in all hypotonic solutions.
The reason is that without energy supply, various membrane pumps (especially Na+-K+ pump) will fail to function. Sodium chloride will enter the cells, they will swell up, and finally rupture.
Q.7 What is the clinical significance of doing fragility test?
Though the fragility test is not done as a routine test, it is employed as a screening test in hereditary spherocytosis.
Q.8 Name the conditions where red cell fragil-ity increases and those where it decreases?
A. Increased red cell fragility is seen in the following conditions:
1. Hereditary spherocytosis. It is one of the commonest causes of hemolytic anemias. A defect in the structural proteins causes them to become spherocytes (in normal plasma), and more fragile. Some red cells are trapped and broken up in the spleen, while others hemolyze in blood.
2. Autoimmune hemolytic anemia. Autoim-mune antibodies damage the structural proteins.
3. Toxic chemicals, poisons, infections, and some drugs (aspirin). These agents make the red cells more fragile in some individuals.
4. Deficiency of glucose 6-phosphate dehydrogenase (G6PD). This enzyme is required for glucose oxidation via hexose monophosphate pathway which generates NADPH. Normal red cells fragility is somehow dependent on NADPH. Deficiency of G6PD, which is the commonest human enzyme abnormality, increases the tendency of the red cells to hemolyze by antimalarial drugs and other agents.
5. The venom of cobra and some insects contains lecithinase which dissolves lecithin
from red cell membranes, thus making them more fragile.
B. Decreased red cell fragility. It is seen in acholuric jaundice and some anemias. The increase in red cell size in pernicious anemia makes them less osmotically fragile as compared to normal red cells (as tested with the above method). However, their mechanical fragility is greater than normal, as a result of which they hemolyze in blood and in spleen.
Q.9 What are the complications of hemolysis occurring in the circulating blood?
The Hb released from red cells will increase the osmotic pressure of blood thereby affecting tissue fluid exchanges. Further, if the tubular fluid is acidic, acid hematin crystals may be precipitated in the renal tubules and cause renal damage.
Q.10 Name some hemolytic agents.
Some of the hemolytic agents are:
1. Hypotonic saline
2. Incompatible blood transfusion 3. Snake venom
4. Severe infection
5. Reaction to certain drugs. Aspirin is a common drug that may cause hemolysis at any time.
Q.11 What is the effect of 5% glucose, 10%
glucose, urea solution of any strength, and urine on red cells?
a. 5% glucose. It is isotonic with blood (and plasma). The RBCs do not show any change in size or shape.
b. 10% glucose. Since it is hypertonic, the red cells will shrink due to exosmosis (water moving out).
However, in the intact body, when 10% or even 20% glucose is given intravenously, the RBCs will shrink in the beginning. But later on, after some time, glucose gets metabolised and there are no harmful effects.
c. Urea solution. As urea tends to move into the red cells, this is followed by water. The final result is hemolysis.
d. Urine. Since urine is hypotonic, the red cells imbibe some water and swell up. In a highly concentrated urine sample, the red cells shrink to some extent.
Q.12 How does hemolysis occur in the body?
Hemolysis of red cells within bloodstream may occur in many different ways. It may be due to structural abnormalities (hereditary spherocytosis, sickle cells), mismatched blood transfusion, bacterial toxins, chemicals, adverse drug reactions, venom of snake and insects.
Q.13 Name some isotonic solutions for mam-mals.
Isotonic solutions of medical interest are:
1. Sodium bromide: 1.5%
2. Magnesium sulphate: 3.3%
3. Sodium chloride: 0.9%
4. Sodium nitrate: 2.5%
5. Dextrose: 5%
6. Sucrose: 10%
7. Sodium bicarbonate: 0.9%
Q.14 Do all the normal RBCs in a person or in a sample of blood have similar osmotic fragility?
The red cells in a person or in a sample of blood vary in their osmotic fragility because they belong to many generations. Younger cells are more resistant while older cells are more osmotically and mechanically fragile (The old and worn out cells fragment in the circulation and are taken up by the RES.) After removal of spleen, the cells become flat which decreases the volume to surface ratio, thereby decreasing osmotic fragility.
STUDENT OBJECTIVES
After completing this experiment, you should be able to:
1. Define specific gravity and indicate the specific gravities of blood, plasma, and serum.
2. Indicate the utility of this test.
3. Name the various fractions of plasma proteins and their functions.
4. List the different lipoprotein fractions and their clinical significance.
Relevance
The “copper sulphate falling drop method” is a rapid and accurate procedure for estimating plasma proteins and hemoglobin concentrations, and hematocrit values in a large number of cases. The test is useful in screening blood donors, and in handling emergency burn cases requiring plasma transfusions. The test is routinely done in many laboratories, and was used extensively during second world war.