Hemolysis is defined as the alteration, dissolution or destruction of red blood cell membranes in such a manner that hemoglobin is freed into the medium in which the cells are suspended. Hemolysis can be caused by antibodies, toxins, chemicals or physical stress from dilution, extremes of pressure, or shear forces or temperature during phlebotomy or preparation of serum. Even time can cause hemolysis, through the exhaustion of available glucose, which is necessary to maintain the fluidity and stability of the cell membranes.
Conceptually, all the mechanisms produce the same ultimate effect by damaging red blood cell membranes and liberating hemoglobin into background liquid, as illustrated here:
Once red blood cell membranes are breached, hemoglobin enters the surrounding media, whether this is serum, plasma, preservatives, other liquids, gels or solids. Compared to serum or plasma, intact red blood cells contain high amounts of certain biologic compounds. In particular, Hemoglobin, which imparts the characteristic red coloration to specimens, and Lactate Dehydrogenase, which has 4000 times as much activity within a red blood cell as there is in the surrounding plasma or serum. Thus, hemolysis to even a small extent releases a large amount of LDH into a serum specimen, altering the native concentration. Thus, hemolysis is a marker of the presence of assay targets that interfere with measurements of these in serum.
In the past, detection of hemolysis in serum specimens was done by eye, which is naturally highly subjective and variable. This led to an uneven application of cancellation criteria of serum Potassium, LDH, Phosphorous and Alkaline Phosphatase results, on the basis of these being higher in red blood cells than the serum. The use of color charts improved consistency somewhat, but still suffered from subjective interpretation, lighting and visual acuity.
Today, modern Chemistry analyzers use discrete wavelengths of light to reflect the amount of hemolysis present. Manufacturers studied each individual target analyte, comparing its scientific precision against how much hemolysis was present. Each target has an assigned hemolysis index, which reflects the amount of hemoglobin present above which the quantitation of that target fails scientific reliability. In other words, if there is too much hemoglobin present for that assay, the test results will be unreliable.
The lowest hemolysis index is for LDH, which makes sense because the amount of this enzyme is 4000 times higher within a red blood cell compared to the serum. Therefore, even a small amount of hemolysis – invisible to the naked eye – alters the serum LDH level to an irregular and unpredictable degree, forcing the cancellation of this target even at low levels unable to be seen visually. In contrast, the hemolysis index of Potassium is much higher, because its concentration inside a red blood cell is much closer to that of serum.
The next in this short series of articles will present practical suggestions on how to avoid hemolysis cancellations, many sites already have few if any cancellations due to hemolysis. In fact, nearly 90% of all specimens fall below the LDH hemolysis index, and your site’s specimens can, too!