Hemolysis refers to the bursting of red blood cells (RBCs), releasing their internal contents, primarily hemoglobin, into the surrounding fluid. The hemolysis assay is a laboratory test designed to measure this. It is significant across scientific and medical fields, providing insights into cellular integrity and potential harmful interactions.
Understanding Hemolysis
Red blood cells, also known as erythrocytes, are disc-shaped cells responsible for transporting oxygen from the lungs to the body’s tissues and carrying carbon dioxide back to the lungs. These cells maintain integrity through their cell membrane, which acts as a barrier. The lifespan of a healthy red blood cell is approximately 120 days, after which they are removed by the spleen.
Hemolysis occurs when this cell membrane is compromised, leading to premature destruction and hemoglobin release into the blood plasma. Various factors can cause this. One common cause is osmotic imbalance, where a hypotonic solution (one with a lower solute concentration than the cell’s interior) causes water to rush into the cell, leading it to swell and burst.
Mechanical stress, such as turbulent blood flow from artificial heart valves or obstructed blood vessels, can physically tear the cell membrane. Toxins produced by certain bacteria or fungi, known as hemolysins, can directly damage the red blood cell membrane, creating holes and inducing lysis. Activation of the complement system, a part of the immune system, can also target and destroy red blood cells, contributing to hemolysis.
Applications of Hemolysis Assays
Hemolysis assays serve many purposes in scientific research and medical diagnostics. A primary application is in drug development and toxicity screening, assessing the potential for new drugs or compounds to damage red blood cells. This is relevant for injectable medications, where the US FDA recommends considering a hemolysis assay for excipients.
The assay is also used to measure the activity of the complement system, an immune response mechanism, in various diseases or conditions. By quantifying complement-mediated hemolysis, researchers can gain insights into immune system dysregulation, for instance, when evaluating nanoparticle biocompatibility for medical use.
While not typically for routine cross-matching, the principle of hemolysis assays is relevant in blood compatibility testing, especially in transfusion medicine, to ensure donor and recipient blood components are compatible. These assays also aid in diagnosing hemolytic anemias, conditions where red blood cells are prematurely destroyed, by identifying the underlying cause of accelerated RBC breakdown.
Beyond clinical applications, hemolysis assays are used in research to study cell membrane integrity, cell-drug interactions, and the effects of environmental factors on blood cells. They provide a straightforward method for preliminary cytotoxicity evaluations of chemicals, drugs, and blood-contacting medical devices or materials.
How Hemolysis Assays Work
Performing a hemolysis assay begins with preparing red blood cell suspensions. Whole blood from a target species (e.g., human, rabbit, or sheep) is collected with an anticoagulant (e.g., EDTA or sodium citrate), then washed to isolate red blood cells. RBC concentration in the suspension commonly ranges from 1% to 4%.
Hemolysis is induced under controlled conditions. This involves exposing red blood cells to a test substance, hypotonic solution, or complement activators, depending on the assay’s objective. The mixture is incubated, often at 37°C for 30 minutes to 24 hours, allowing the test substance to interact with the red blood cells.
After incubation, samples are centrifuged to separate unlysed red blood cells from the supernatant, which contains released hemoglobin. Hemoglobin in the supernatant is then measured using a spectrophotometer. Hemoglobin absorbs light at specific wavelengths (e.g., 415 nm or 540 nm), allowing for precise quantification based on light intensity.
Accurate results require both positive and negative controls. A negative control (e.g., assay buffer) represents no hemolysis, while a positive control (e.g., lysis fluid or Triton X-100) induces complete red blood cell lysis, providing a reference for 100% hemolysis. These controls normalize results and account for background interference or experimental variations.
Interpreting Hemolysis Assay Results
Hemolysis assay results are expressed as a percentage, indicating the proportion of red blood cells lysed relative to a complete lysis control. This percentage is calculated by comparing hemoglobin released in the test sample to total hemoglobin from a fully lysed sample.
Researchers generate dose-response curves by testing varying concentrations of a substance. This allows determination of the effective concentration (EC50), the concentration causing 50% hemolysis. These curves provide insight into the substance’s potency and the relationship between concentration and red blood cell destruction.
High hemolysis levels can indicate toxicity of a tested compound to red blood cells or elevated immune activity. Conversely, low levels suggest a substance is less likely to cause red blood cell damage or that immune activation is within expected ranges. For instance, in pharmaceutical formulations, hemolysis values below 10% are generally considered non-hemolytic, while values above 25% indicate a risk of hemolysis.
Limitations must be considered when interpreting hemolysis assay results, as various factors can influence the outcome. Blood source species can affect hemolytic response, with differences up to fourfold between species. Variations in erythrocyte concentration, incubation time, and positive control detergent type can significantly impact measured hemolysis, highlighting the need for standardized protocols for reliable data interpretation.