When a sample is hemolyzed, why is it important to distinguish in vivo from in vitro hemolysis?

In vivo hemolysis happens due to numerous biochemical, physical, chemical, and immunological mechanisms, and/or infections that occur within the body prior to blood being drawn. Correctly identifying it is of great clinical importance because it is a sign of many different underlying pathological conditions, some of which could be life-threatening if left untreated.

Potential causes include: 1) autoimmune hemolytic anemia and hemolysis following incompatible blood transfusion; 2) intrinsic red blood cell defects (e.g., hemoglobinopathies, thalassemias, and various enzyme defects such as glucose-6-phosphate dehydrogenase deficiency and pyruvate kinase deficiency); 3) mechanical hemolysis (e.g., from a mechanical heart valve); and 4) microangiopathic hemolysis (e.g., disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome).

In vivo hemolysis can be further sub-characterized into intravascular or extravascular hemolysis depending on the mechanism and site of red blood cell destruction. In vitro hemolysis, on the other hand, occurs outside of the body and is most often the result of preanalytical factors such as blood drawing, specimen handling, specimen delivery to the laboratory, or specimen storage.

How does hemolysis affect laboratory tests?

The most common effects of hemolysis on chemistry tests include: 1) increases in analyte concentration due to the release of red blood cell constituents (affected analytes include potassium, magnesium, aspartate aminotransferase, and lactate dehydrogenase [LDH]); 2) increases in analyte concentration due to assay interference (affected analytes include cholesterol, triglycerides, and creatinine kinase); and 3) decreases in analyte concentration due to assay interference (affected analytes include bilirubin, insulin, and albumin).

What are the pros and cons of using haptoglobin as an indicator of in vivo hemolysis?

Decreased concentrations of haptoglobin in serum and free hemoglobin in urine are the most pronounced laboratory indicators of in vivo hemolysis. In more severe cases haptoglobin may be undetectable, whereas with in vitro hemolysis the concentration of haptoglobin is usually not affected. However, few studies have examined the validity of haptoglobin as a marker of in vivo hemolysis. The studies that do exist have shown varying degrees of sensitivities and specificities for haptoglobin depending on the cutoff concentration used.

One used a haptoglobin cutoff of less than 25 mg/dL and demonstrated 83% sensitivity and 96% specificity for intravascular hemolysis (JAMA 1980;243:1909-11). Another used a haptoglobin cutoff of less than 28 mg/dL and noted 91.8% sensitivity and 98.4% specificity for intravascular hemolysis (Eur J Clin Invest 2006;36:202-9).

In general, there is no gold standard test to confirm in vivo hemolysis, and most often labs rely on other clinical factors (e.g., increased reticulocyte count) and correlation with other laboratory markers (e.g., complete blood count, LDH, and indirect bilirubin) in addition to a patient’s history.

What other clinical factors influence the concentration of haptoglobin?

A variety of factors aside from hemolysis influence haptoglobin concentrations. For example, haptoglobin is often considered to be a nonspecific acute phase reactant and elevated haptoglobin concentrations occur in many inflammatory conditions, including severe infection, inflammation, tissue destruction, acute myocardial infarction, burns, and some cancers.

On the other hand, haptoglobin concentrations can also decrease in the absence of hemolysis. Pathological conditions associated with decreased haptoglobin concentrations include liver cirrhosis, pulmonary sarcoidosis, and elevated estrogen states.

Congenital anhaptoglobinemia is a relatively benign condition that results in the absence of haptoglobin. In addition, hemodilution and blood transfusions can lead to falsely reduced haptoglobin concentrations. Differentiating hemolysis from all these conditions is difficult given that both intravascular and extravascular hemolysis can cause reduced haptoglobin concentrations.

Consequently, clinicians as well as laboratorians need to be aware of haptoglobin’s limitations and interactions, and interpret this test in the context of a patient’s clinical scenario.

John V. Mitsios, PhD, is assistant director of the special coagulation laboratory at BioReference Laboratories in Elmwood Park, New Jersey. +Email: jmitsios@bioreference.com