HAPTOGLOBIN Educational Commentary Is Provided Through

EDUCATIONAL COMMENTARY – HAPTOGLOBIN

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Learning Outcomes Upon completion of this exercise, the participant will be able to: • explain the function of haptoglobin. • discuss the conditions that result in a decreased haptoglobin level. • describe intravascular and extravascular red blood cell destruction. • define acute-phase protein. • discuss the available methods to test for haptoglobin level.

Haptoglobin is a major protein produced in the liver that plays an important role in preventing iron loss from the body. Hemoglobin is the protein in the red blood cell (RBC) that contains iron and is responsible for binding oxygen from the lungs and carrying it to the tissues throughout the body. The function of haptoglobin is to bind to free hemoglobin and form haptoglobin-hemoglobin complexes. These complexes are transported to the liver where hepatocytes (liver cells) degrade the complexes into their components: hemoglobin and haptoglobin. During this process iron is retained and recycled.

Intravascular and Extravascular RBC Destruction Only small amounts of free hemoglobin are found in the circulation unless the person has a condition in which RBC destruction is increased. There are two types of RBC destruction: intravascular and extravascular. Haptoglobin concentration can help differentiate the two types of RBC destruction. Normal destruction of RBCs occurs as the RBCs reach the end of their 120-day life span. Macrophages in the spleen engulf the RBCs and remove them from the circulation. This is called “extravascular erythrocyte destruction.” Free hemoglobin is not released into the circulation and haptoglobin levels remain normal. Ninety percent of aged RBCs are destroyed in this manner. In increased intravascular RBC destruction, RBCs are lysed in the bloodstream and hemoglobin is released. Individuals with intravascular hemolytic anemia have decreased haptoglobin levels because so much hemoglobin is released from the RBCs that most of the circulating free haptoglobin binds to the free hemoglobin. The liver is unable to produce haptoglobin rapidly enough to maintain the normal concentration of haptoglobin in the bloodstream. Usually low haptoglobin levels indicate hemolytic anemia. Causes of hemolytic anemia include disorders such as autoimmune hemolytic anemia, hemolytic disease of the newborn, sickle cell anemia, thalassemia, and blood transfusion reactions. Decreased haptoglobin in the absence of hemolytic anemia may indicate inadequate production of haptoglobin by the liver.

American Proficiency Institute – 2010 2nd Test Event EDUCATIONAL COMMENTARY – HAPTOGLOBIN (cont.)

Acute-Phase Proteins Haptoglobin is one of the proteins known as an acute-phase reactant. These proteins increase in concentration during an inflammatory response such as rheumatoid arthritis, bacterial or viral infections, burns, or trauma. During inflammation, synthesis of acute-phase plasma proteins increases in the liver. The function of acute-phase proteins is to help remove cells and tissue damaged during an inflammatory process. They play a significant role in wound healing. Haptoglobin concentrations can increase two to four times the normal range when inflammation occurs. Increased haptoglobin concentrations can also be caused by massive blood loss, kidney disease, or liver disease.

Assaying Haptoglobin A physician may order a haptoglobin analysis for patients who show signs of anemia, such as pallor, shortness of breath, jaundice, and fatigue. Haptoglobin is not used alone in the diagnosis of hemolytic anemia. Other tests that may be ordered in conjunction with the haptoglobin level include a complete blood cell count with differential, reticulocyte count, bilirubin level, and a direct antiglobulin test if a transfusion reaction or autoimmune anemia is suspected.

Various assay methods are used in haptoglobin determination. In the immunoturbidimetric assay, haptoglobin in the patient serum combines with reagent anti-haptoglobin to form antigen-antibody immune complexes. The formation of the immune complexes increases the turbidity (cloudiness) of the solution. A spectrophotometer measures absorbance. Changes in absorbance over time may indicate the presence of the immune complexes. The absorbance change is compared with that of a standard (calibrator) of known concentration. The greater the absorbance change, the higher the haptoglobin level.

Nephelometry is another way to measure haptoglobin. Anti-haptoglobin antibodies bind with circulating haptoglobin to form immune complexes as in immunoturbidimetry. Incident light passes through the solution and is reflected when it hits the immune complexes. The reflected light is measured by a detector. The amount of reflected light from the patient sample is compared with a standard curve. The more reflected light, the higher the concentration of haptoglobin.

Enzyme-linked immunosorbent assay (ELISA) is also used to measure haptoglobin. Microtiter wells are coated with anti-haptoglobin antibody. Standards, controls, and patient sera are added to the wells and allowed to incubate. If haptoglobin is present, it forms a complex with the antibody coated on the well. A washing procedure is performed to remove all unbound substances. The next reagent is a conjugate composed of another anti-haptoglobin antibody with an attached enzyme. This antibody will bind to a different site on the haptoglobin molecule. After washing again, an enzyme substrate is added, and the microtiter plate is incubated. If haptoglobin is present in a well, the substrate will degrade and a color will

American Proficiency Institute – 2010 2nd Test Event EDUCATIONAL COMMENTARY – HAPTOGLOBIN (cont.) develop. Absorbance of each well is read on a spectrophotometer. The concentration of haptoglobin in a well is directly proportional to the color produced.

Conclusion Intravascular hemolysis is characterized by markedly decreased haptoglobin levels with anemia, hemoglobinuria, and increasing total bilirubin. Conversely, extravascular hemolysis is characterized by anemia and jaundice, with a normal or mildly decreased haptoglobin level. Therefore, a low haptoglobin, a low RBC count, a low hemoglobin, and a low hematocrit, with an increased reticulocyte count, can suggest the presence of a hemolytic anemia. On the other hand, a normal haptoglobin level with an increased reticulocyte count, in a patient with a suspected hemolytic anemia, may indicate extravascular RBC destruction. Hemoglobin is not released from the RBC into the bloodstream, and haptoglobin is not bound. Therefore, the haptoglobin level would remain normal. A low haptoglobin concentration without signs of anemia indicates an inadequate production of haptoglobin by the liver.

Suggested Reading Haptoglobin. Lab Tests Online. Available at http://www.labtestsonline.org/understanding/analytes/haptoglobin. Accessed on May 19, 2010.

American Proficiency Institute – 2010 2nd Test Event