Figure 1. Red Cell Changes in Hemolytic Disorders. A defining feature of hemolytic disorders is a decrease in red cell survival. Hereditary hemolytic disorders arise as a consequence of intrinsic defects in genes/proteins expressed during RBC development. For many disorders with accelerated red cell turnover, and for normal RBC senescence, the sequence of events and nature of the signal triggering removal from circulation by reticuloendothelial cells (primarily macrophages in the spleen) remains unclear. When morphologic changes in the RBC are seen, this suggests immune-mediated damage, hemoglobinopathy or a membrane defect. Many red cell enzyme defects shorten RBC survival, but do not alter the appearance of the red cell. (RBC = red blood cell)

What are some of the different types of hemolytic anemia?

One way to categorize hemolytic anemia is to separate hereditary anemia from acquired anemia. An example of an acquired hemolytic anemia would be the red cell destruction that occurs during malaria infection. Causes of acquired hemolytic anemia include infection, drug or toxin exposure and autoimmune reaction against red cells. Hereditary hemolytic anemia implies an underlying gene change or mutation is responsible for red cell destruction.

Figure 2. Red Cell Development and Hemolytic Disorders. Hereditary hemolytic disorders arise as a consequence of intrinsic defects in genes/proteins expressed during RBC development. When severe, such defects can result in increased marrow turnover of immature cells (ineffective erythropoiesis), in addition to premature destruction of RBC in the circulation. Extrinsic factors can also reduce red cell survival and cause acquired forms of hemolytic anemia. Infection, immune destruction of red cells and exposure to certain drugs or toxins are examples of extrinsic causes of hemolytic anemia.

Some types of hereditary hemolytic anemia are well described—for instance sickle cell anemia (Gene Reviews/Genetics Home Reference) and thalassemia (Genetics Home Reference [alpha]/Genetics Home Reference [beta]) due to changes affecting the sequence or activity of the hemoglobin genes, and G6PD (glucose-6-phosphate dehydrogenase) deficiency which affects the activity of a key red cell enzyme. Genetic causes for several other types of hereditary hemolytic anemia have been described—and will be discussed below. In many cases of hereditary hemolytic anemia the genetic cause remains unknown, and the discovery of new genes that play a role in this disorder is an area of active research. Much of the rest of this discussion focuses upon hereditary hemolytic anemia caused by red cell enzyme defects or red cell membrane protein defects, as well as ongoing research to discover new genes responsible for currently undefined hereditary anemia. For a more general overview of hemolytic anemia, including acquired disorders, the National Heart, Lung and Blood Institute maintains this helpful web site: http://www.nhlbi.nih.gov/health/dci/Diseases/ha/ha_whatis.html.

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What causes hemolytic anemia?

A shortened red cell survival is a symptom, not a cause of hemolytic anemia. How is red cell survival measured? The accompanying figure shows idealized red cell survival curves using two distinct methods of red cell labeling (cohort labeling or random labeling). In each case, normal red cells persist in the circulation longer than cells from a patient with a hemolytic disorder. In practice, physicians rarely directly measure red survival. Instead, they rely upon indirect indices of red cell survival derived from the complete blood count (CBC), the reticulocyte count (fraction of immature red cells), examination of the bone marrow, and blood chemistry to estimate the rate of red cell turnover.

Once a patient is diagnosed with hemolytic anemia, a series of laboratory evaluations is performed to rule in or rule out specific known causes. These evaluations may include all or a subset of the following tests:

1. Blood smear—a sample of peripheral blood is examined microscopically. Abnormalities in the size, shape or staining pattern of red cells can provide clues as to the type of disorder causing red cell destruction.

2. Coombs test—determines whether an immune reaction is leading to blood cell destruction. For more information, click the following National Institute of Health link: http://www.nlm.nih.gov/medlineplus/ency/article/003344.htm

3. Hemoglobin electrophoresis and test for unstable hemoglobin are performed to determine whether there are abnormalities in the hemoglobin protein or an imbalance of hemoglobin production. For more information, click the following National Institute of Health link: http://www.nlm.nih.gov/medlineplus/ency/article/003639.htm

4. Osmotic fragility test is performed if a defect in the red cell membrane is suspected. This test measures the integrity or strength of the red cell membrane by placing cells into a test solution that is more dilute than human plasma. Normal and diseased cells will swell and rupture, but cells with membrane defects rupture sooner. For more information, click the following National Institute of Health link: http://www.nlm.nih.gov/medlineplus/ency/article/003641.htm

5. Red cell enzyme assay panel is performed when an enzyme defect is suspected and/or the above tests are normal. The most common red cell enzyme defects are glucose-6-phosphate dehydrogenase deficiency and pyruvate kinase deficiency—although several other enzyme defects have been described (again, link to table with more detailed listing cited in the ‘What causes section’). For a more complete list of enzyme defects associated with hereditary hemolytic anemia, click here.

What are some of the problems that predispose red cells to premature destruction? The short answer is that we don’t know all the problems that result in premature red cell destruction. One common theme is that premature red cell loss is often (but not always) associated with a breakdown in red cell defenses against oxidative damage.

Figure 4.  Pathways Protecting RBC from Oxidative Damage.  The red cell possesses an overlapping series of reactive oxygen species (ROS) detoxifying enzymes—particularly for disposing of peroxides.  In mice, knockout of catalase or glutathione peroxidase (GPx1) has little impact on red cells under normal conditions, while knockout of superoxide dismutase 1 (SOD1) or peroxiredoxin 2 (PRDX2) results in hemolytic anemia.  The activity of ROS detoxifying enzymes is dependent upon chemical reductants (NADPH or NADH) derived from major red cell metabolic pathways--the hexosemonophosphate (HMP) pathway and the glycolytic pathway.  Defects in these pathways that interfere with production of NADPH, NADH or the energy carrier ATP can also cause hemolytic anemia.

Much of red blood cell metabolic activity is devoted to protecting against or repairing ongoing oxidative damage. The figure above lists several enzymes that play a part in protecting red cells against oxidative damage. Directly or indirectly, several of these enzymes utilize cofactors such as the antioxidant (reduced) glutathione, NADPH or NADH in chemical reactions to detoxify ROS or reverse oxidative damage to red cell proteins. Gene defects that interfere with red cell production of (reduced) glutathione, NADPH or NADH are recognized as causes of hereditary hemolytic anemia.

Figure 5. Major RBC Metabolic Pathways. Reductive power for both GPx and PRDX is ultimately derived from NADPH produced in the hexosemonophosphate (HMP) pathway. GPx oxidizes glutathione, which is regenerated by glutathione reductase (GRx) in an NADPH coupled reaction. Oxidized PRDX is reduced by thioredoxin (Trx), which is in turn reduced by thioredoxin reductase (TrRx) in an NADPH coupled reaction. Methemoglobin is reduced by cytochrome b5 reductase (cytb 5) in a reaction utilizing NADH generated during glycolysis. These represent the major RBC pathways for reversing ongoing oxidative damage.

Among RBC metabolic enzymes, common, rare and extremely rare mutations that cause hemolytic anemia have been characterized. The most common mutations occur in the gene encoding glucose-6-phosphate dehydrogenase (G6PD). Less common are mutations in the gene encoding pyruvate kinase (PK). More rare still are mutations affecting the genes encoding glucose-phosphate isomerase (GPI), adenylate kinase (AK) and cytochrome b5 reductase (cytb5-R). For a more complete list of enzyme defects associated with hereditary hemolytic anemia, click here.

In addition to the hemoglobin and enzyme defects noted above, problems with red cell scaffold proteins can affect the size, shape and mechanical properties of the RBC, leading to premature destruction. Collectively, these defects are described as red cell membrane disorders. As with the enzymatic defects, a number of gene mutations have been described that cause hemolytic anemia in which the red cells assume characteristic shapes—hereditary acanthocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis, hereditary spherocytosis and hereditary stomatocytosis. For more information on red cell membrane proteins, click here.

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How is (hereditary) hemolytic anemia treated?

In general, hereditary hemolytic anemia is treated with supportive care that includes ensuring patients have adequate nutrition to support red cell production. In severe cases, patients are treated with red cell transfusions as needed to maintain their red cell count. Some types of hemolytic anemia can be improved with splenectomy--but this decision must be carefully considered as removal of the spleen places patients at increased risk for infection. Bone marrow transplantation is a potentially curative option for severe hereditary hemolytic anemia, and has been used successfully in treatment of thalassemia and sickle cell disease. Marrow transplantation carries significant risks, and it is often difficult to find a suitable donor—consequently this therapy remains uncommon even in severe hemolytic anemia cases.

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What research is being done to learn about the causes of hemolytic anemia?

While a great deal has been learned about causes of hereditary hemolytic anemia, it is not unusual for patients/families with this disorder to find out that no recognized gene defect can explain the disorder that runs in THEIR family. By some estimates, more than half of patients with hereditary hemolytic anemia find no cause for their disorder using currently available diagnostic methods. It is for this reason that the National Institutes of Health (NIH) through the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) supports basic research to identify the causes of hemolytic anemia. This website is funded by the NIDDK as an education and outreach component of a basic research project into the causes of hereditary hemolytic anemia. The home base for this project is the Friedman laboratory at the Scripps Research Institute in La Jolla, California. We thank patients, their families and referring physicians for their participation in this project aimed at identifying new causes of hemolytic anemia. Below is a description of the major goals and rationale behind our search for new causes of hemolytic anemia. For a more detailed description of our science, you may refer to the ‘for physicians’ page on this website, or contact our laboratory at the Scripps Research Institute.

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Research Description:

Our laboratory explores new approaches to aid the diagnosis and evaluation of hereditary hemolytic anemia. Known causes of these disorders include mutation in genes encoding several red cell enzymes and red cell structural proteins. Diagnostic approaches are imperfect because often no ‘lesion’ (explanation or cause for hemolysis) is found, and current diagnostic methods are both expensive and complicated.

The NIH has funded our lab to explore the use of an unbiased proteomic approach to pinpoint proteins that are present in abnormal amounts (new proteins, or loss of expected proteins) in patients without a molecular diagnosis. The picture below provides an example of how proteins are measured and compared.

Figure 6. 2 Dimensional Gel Comparison of Red Cell Proteins: This figure provides an example of a gel electrophoresis of red cell proteins from a hemolytic anemia patient, the patient’s mother, and a control sample using a sensitive fluorescent method called DIGE.

We use a specialized ‘scanner’ to take accurate pictures of the proteins in our gels, and then use image analysis software to match the protein spots in each picture, and determine whether the amount of a specific protein is different in the patient’s red cells compared to normal. An example of this type of analysis is shown below.

Figure 7. Two Examples of Proteins Altered in Hemolytic Anemia: In this example, red blood cell proteins from two hemolytic anemia patients were compared against their unaffected parents. In part A is shown a protein that is elevated in both patient samples; in part B is shown a protein that is decreased in both patients.

The next step is to identify the protein ‘spots’ that differ between normal and anemic samples. Once proteins are identified, there are many potential ways to evaluate whether a change in abundance of a specific protein is related to development of hemolytic anemia. If we are successful, there are two ways this work will have practical impact: First, identification of protein alterations related to development of hemolytic anemia will facilitate development of diagnostic tests that can be applied to existing and new patients with hemolytic anemia. Ultimately, such tests may be useful to define carrier status or for purposes of prenatal diagnosis. Second, by identifying the genes responsible for hemolytic anemia, it may be possible to design rational therapies for specific types of hemolytic anemia.

Another aspect of our study goes back to Figure 4 above—a focus on oxidative damage as a factor in development of this type of anemia. To study this process, we have developed assays to measure levels of reactive oxygen species (ROS) in red cells, and to measure oxidative damage to red cell proteins. We hypothesize that many different types of hemolytic anemia (but not all) result from defects in the ability of the red cell to protect itself against oxidative damage. Thus far, we have found an increase in ROS in red cells from many ‘undiagnosed’ hemolytic anemia samples. This suggests to us that interventions that decrease reactive oxygen species or that protect against oxidative damage may be beneficial in some patients with hemolytic disorders. We are testing this idea in mouse models of hemolytic anemia.

If you would like to learn more about hemolytic anemia research, have questions about our research project, or have questions about participation in this research, we welcome your inquiries. To contact us, click here.

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