Hemolysis occurs when red blood cells (RBCs) rupture, releasing internal components, primarily hemoglobin, into the surrounding blood plasma or serum. This process changes the sample’s integrity, making it unsuitable for accurate laboratory analysis. To standardize the detection and quantification of this issue, laboratories use the Hemolysis Index (HI). This measurement acts as a quality control checkpoint, allowing professionals to determine objectively if a blood sample is reliable for diagnostic testing. An elevated index signals a potential problem that could lead to inaccurate patient results, underscoring its importance in medical diagnostics.
Defining the Hemolysis Index
The Hemolysis Index is a quantitative measurement, often referred to as the H-index or H-score, designed to assess the concentration of free hemoglobin in a blood specimen. Modern chemistry analyzers measure this index automatically using a technique called spectrophotometry. This method works by shining light through the plasma or serum and measuring how much is absorbed by the red-colored free hemoglobin.
Hemoglobin absorbs light at specific wavelengths. The index calculates the degree of hemolysis by measuring the absorbance at these points. The resulting value is typically reported on a semi-quantitative scale (e.g., 1+ to 4+) or as a numeric unit correlating to a specific concentration of free hemoglobin, such as milligrams per deciliter (mg/dL). A higher value indicates a more severely hemolyzed sample. Laboratories establish specific cutoff points for the index, above which the sample is considered compromised and unsuitable for certain tests.
Differentiating Causes of Hemolysis
Hemolysis occurs due to two primary reasons: factors outside the body related to sample collection and handling, or conditions inside the body related to a patient’s disease state. The vast majority of elevated hemolysis indices (40% to 70% of unsuitable specimens) are caused by factors outside the body, known as in vitro hemolysis. This type of hemolysis is a pre-analytical error that occurs after the blood leaves the patient’s vein.
Pre-analytical errors mechanically damage the fragile cell membranes. These errors include:
- Using an incorrect or too-small needle size, which forces RBCs to break apart.
- Applying excessive suction during venipuncture or vigorously shaking the collection tube.
- Delayed processing of the sample or jarring transportation.
- Exposure to temperature extremes like freezing or excessive heat.
While in vitro errors are the most common cause, hemolysis can also occur in vivo (within the patient’s body). This happens in pathological conditions such as inherited or acquired hemolytic anemias, certain autoimmune conditions, or severe infections. In these cases, the elevated index accurately reflects the patient’s underlying medical condition. However, the laboratory primarily uses the index to flag sample quality, and a high reading usually requires ruling out a handling error first.
Impact on Laboratory Results
Hemolysis interferes with test results through two mechanisms: the release of highly concentrated intracellular substances and the optical interference caused by the free hemoglobin itself. The release of components from inside the ruptured red blood cells into the plasma significantly skews the measured concentration of various analytes.
One clinically significant interference is the false elevation of potassium, known as pseudohyperkalemia. Red blood cells maintain an intracellular potassium concentration that is roughly 25 times higher than the normal plasma concentration. When RBCs rupture, this concentrated potassium floods the plasma, leading to a falsely high test result that does not reflect the true level in the patient’s circulation.
The levels of certain enzymes are also dramatically affected because they are highly concentrated inside RBCs. For example, Lactate Dehydrogenase (LDH) and Aspartate Aminotransferase (AST) are released, causing a false increase in their measured activity. Since high levels of potassium, LDH, and AST are often used to diagnose serious conditions, such as heart attack or liver damage, a hemolyzed sample can lead to misdiagnosis or unnecessary treatment.
The second mechanism of interference is analytical. The red color of free hemoglobin absorbs light in the same range as the chemicals used in many laboratory assays. This optical interference can lead to falsely high or falsely low results for analytes such as Bilirubin. Because the interference is unpredictable, a high hemolysis index typically invalidates the sample for sensitive tests. The laboratory must reject significantly elevated samples and request a re-draw to obtain accurate data.