Collecting a blood sample is often the first step in a medical diagnosis. A common problem encountered in the clinical laboratory is the rejection of a blood specimen due to hemolysis. Hemolysis is the rupture, or lysis, of red blood cells (RBCs), which releases their internal contents into the surrounding plasma or serum. This rejection is a safety measure to ensure accurate test results, as a hemolyzed sample can lead to misdiagnosis or inappropriate treatment. While hemolysis can occur inside the body due to a medical condition, most rejected samples are hemolyzed outside the body during collection or handling.
What Hemolysis Looks Like
Hemolysis occurs when the red blood cell membrane is damaged, causing the cell to break open and spill its contents. The most abundant substance released is hemoglobin, the protein that gives blood its red color. Normally, the liquid portion of the blood (serum or plasma) is a clear, pale yellow. When red blood cells rupture, the free hemoglobin floods the plasma, causing the color to shift from a faint pink tint to a deep, dark red in severely damaged specimens. Even levels not easily seen visually can interfere with testing, which is why modern analyzers often use photometric checks to quantify free hemoglobin.
Physical and Chemical Interference with Assays
The presence of free hemoglobin and other intracellular components interferes with laboratory tests through two mechanisms: physical and chemical interference. Both mechanisms can lead to erroneous results, making the sample unreliable for analysis. Because the accuracy of a test result depends on the quality of the specimen, the lab must often reject the sample.
Physical interference is caused by the light-absorbing properties of hemoglobin. Many clinical chemistry tests use photometry, which measures how much light a sample absorbs at specific wavelengths to determine substance concentration. Hemoglobin strongly absorbs light, particularly between 340–580 nanometers. This intense absorption can overlap with the wavelengths used to measure other analytes, causing the instrument to read a falsely high or low concentration.
Chemical interference results from the release of substances normally contained within the red blood cells into the plasma. Red blood cells have a significantly different internal composition than the surrounding plasma. When they rupture, the released components contaminate the plasma, altering the concentration of analytes in the sample. Furthermore, some released intracellular components, such as certain enzymes, can chemically react with the reagents used in the test, skewing the final result.
Specific Impact on Common Laboratory Tests
The effect of hemolysis is not uniform across all tests; some analytes are highly sensitive to intracellular contamination. Potassium (K+) testing is highly affected by hemolysis. Red blood cells contain a potassium concentration about 25 times higher than what is found in the plasma. When cells rupture, this large amount of potassium spills out, leading to a falsely elevated result, a condition called pseudohyperkalemia.
Enzymes such as Lactate Dehydrogenase (LDH) and Aspartate Aminotransferase (AST) are present in much higher concentrations inside red blood cells than in the plasma. Hemolysis releases these enzymes, causing their measured levels to be artificially high. This could be mistakenly interpreted as evidence of tissue damage or liver injury, as even mild hemolysis can significantly interfere with their results.
Hemolysis can also interfere with other tests, sometimes causing falsely decreased results. For example, released hemoglobin can interfere with the chemical reaction used to measure bilirubin, leading to a falsely low reading. Coagulation tests, such as the Activated Partial Thromboplastin Time (aPTT), can also be interfered with. This interference is due to the presence of cellular debris and released proteases from the ruptured cells.
Avoiding Hemolysis During Collection and Processing
Preventing hemolysis requires careful technique during the pre-analytical phase, which covers collection and initial handling. Errors typically occur during the collection technique or the subsequent sample processing.
Collection Technique
Using a needle smaller than 23 gauge creates excessive shear stress on the red blood cells as they are drawn. Pulling too quickly on the syringe plunger can also generate destructive pressure that ruptures the cells.
Sample Processing
Mishandling the sample after collection can cause hemolysis. Vigorously shaking or mixing the blood tubes, instead of gently inverting them, subjects the cells to physical trauma. Not allowing the alcohol used to clean the venipuncture site to fully air dry can destroy the cell membranes. Under-filling tubes containing an anticoagulant promotes cell rupture due to an incorrect blood-to-additive ratio.