Definition Red Cell Fragility Test. Principle, Procedure and Clinical Significance of Red Cell Fragility Test.

• Red Cell Fragility (Erythrocyte Fragility):

Definitions

• 1. Red Cell Fragility (Erythrocyte Fragility): The tendency of erythrocytes to hemolysis (rupture/break down) under stress is called red cell fragility.

• 2. Red cell fragility test (Erythrocyte Fragility Test): The test to measure erythrocyte fragility resistance under stress is called the Red Cell Fragility Test. The Red Cells Osmotic Fragility test is used to test red cell fragility.

• 3. Isotonic Solutions: An aqueous solution with a solute concentration similar to body fluid solute concentration is called an isotonic solution. Solute concentration inside blood cells and blood plasma is the same. Thus plasma is an isotonic solution. Some common examples of isotonic solutions are 0.9% w/v sodium chloride solution, 5% w/v dextrose solution, blood plasma, etc. The osmotic pressure of isotonic solution and body fluid is the same. Thus, the isotonic solution is also called an iso-osmotic solution. Blood plasma is an isotonic solution. Thus, fluid does not move either into blood cells from blood plasma or out from blood cells into blood plasma.

• 4. Hypertonic solution: An aqueous solution with a solute concentration more than the solute concentration of body fluid is called a hypertonic solution. If a hypertonic solution surrounds blood cells, then fluid moves out from the blood cells. Blood cells shall shrink.

• 5. Hypotonic solution: An aqueous solution with a solute concentration less than the solute concentration of body fluid is called a hypotonic solution. If a hypotonic solution surrounds blood cells, then fluid moves into the blood cells from the hypotonic solution. Blood cells shall swell. Blood cells may burst due to excessive swelling. This is the death of blood cells. Thus hypotonic solution is more dangerous than hypertonic solution.

• 6. Hemolysis: The breakdown of erythrocyte cells is called hemolysis.

• 7. Osmotic fragility tests: Osmotic fragility test measures the resistance of red blood cells (RBCs or erythrocytes) to hemolysis (rupture) in solutions of various levels of dilute solution (Various levels of hypotonic solution). This test is used to diagnose conditions that affect the structure or function of RBC membranes.

Dr Pramila Singh

Red Cell Fragility Test ((Erythrocyte Fragility)

Following two types of fragility tests are used to find out erythrocytes' fragility

• 1. Osmotic erythrocyte fragility test

• 2. Mechanical erythrocyte fragility test

Osmotic erythrocyte fragility test: Principle, Procedure and Clinical Significance.

Principle:

• It is also known as the Red Cell Osmotic Fragility Test or Red Cell Fragility Test. It measures erythrocytes' (Red Blood Cells) resistance to hemolysis (rupture) on exposure to dilute saline solutions (Various levels of hypotonic solution).

• Red Blood Cells (Erythrocytes) maintain their structure in isotonic solutions (Solutions with the same osmotic pressure as the blood).

• Inside the hypotonic solution, water enters from the hypotonic solution into red blood cells. This causes swelling in red blood cells. It may rupture red blood cells and release hemoglobin into surrounding fluid. Rupturing of RBCs is called hemolysis. Hemolysis is assessed by measuring hemoglobin present in fluid surrounding hemolysed cells. The degree of hemolysis is directly proportional to the fragility of the red blood cell membrane.

Procedure:

• 1. Blood Sample Collection: Collect venous blood samples from the patient.

• 2. Preparation of Saline Solutions: Prepare a series of saline hypotonic solutions with decreasing salt concentrations.

• 3. Incubation: Divide the blood sample into multiple tubes. Dilute the blood sample in each tube with the hypotonic saline solutions. Incubate these samples at 37°C for 30 minutes.

• 4. Centrifugation: Centrifuge the incubated samples to separate the lysed RBCs from the intact RBCs.

• 5. Hemolysis Measurement: Measure the degree of hemolysis in each saline hypotonic solution by determining the amount of hemoglobin in the supernatant. This is done by spectrophotometer.

• 6. Interpretation: The results are plotted on a graph. The salt concentration on the x-axis and the percentage hemolysis on the y-axis. This helps to plot the osmotic fragility curve (hemolysis curve). This shows the range of salt concentrations at which hemolysis occurs.

  • i. Normal Results: Most RBCs are resistant to hemolysis in isotonic solutions. It begins to lyse in progressively more hypotonic solutions.

  • ii. Increased Fragility: Hemolysis occurs at higher salt concentrations. It indicates that the RBCs are more fragile than normal.

  • iii. Decreased Fragility: Hemolysis occurs at lower salt concentrations. It suggests that the RBCs are less fragile than normal.

Clinical Significance: The red cell fragility test is to diagnose, monitor, and understand hematological disorders that affect the structure and functions of RBCs. Examples are hemolytic anemia, Sickle cell anemia, thalassemia, drug-induced hemolysis, blood storage conditions evaluation, etc.

Increased Red Cell Fragility:

• Hereditary Spherocytosis: Production of spherical RBCs. These RBCs have decreased surface area. They are more susceptible to hemolysis.

• Autoimmune Hemolytic Anemia: The immune system damages RBCs. It increases RBC fragility.

• Certain Drug-Induced Hemolytic Anemias: Some drugs can damage the RBC membrane. This causes increased fragility.

Decreased Red Cell Fragility:

• Sickle Cell Anemia: Abnormal hemoglobin polymerization within RBCs occurs in sickle cell anemia. This leads to increased cell rigidity and decreased fragility.

• Thalassemia: Blood disorders reduce hemoglobin production. This affects RBC membrane stability.

Limitations:

1. The test is not specific for any particular disease.

2. Other factors, such as temperature and pH, can influence the test results.

3. The test may not be sensitive enough to detect mild abnormalities in RBC fragility.

Dr Pramila Singh