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The autoimmune hemolytic anemias (AIHA) are rare but important hematologic diseases. They can range in severity from mildly symptomatic illness to a rapidly fatal syndrome. The incidence of AIHA is estimated to be between 0.6 and 3 cases per 100,000 persons.1,2 AIHA is mediated by antibodies, and in the majority of cases immunoglobulin (Ig) G is the mediating antibody. This type of AIHA is referred to as "warm" AIHA because IgG antibodies bind best at body temperature. "Cold" AIHA is mediated by IgM antibodies, which bind maximally at temperatures below 37°C (Table 1). This article series reviews the most common types of AIHA, with an overview of evaluation and diagnosis presented in this article and management of warm, cold, and drug-induced AIHA reviewed in a separate article.
Pathogenesis
In most cases, the ultimate etiology of AIHA is unknown. In warm AIHA, the target epitopes in most cases are Rh proteins.2 What leads the immune system to target these proteins is unidentified, but one theory is that an initial immune response to a foreign antigen starts to cross-react with the Rh proteins and the immune system fails to suppress this autoreactive response, leading to hemolysis. In IgG-mediated (warm) hemolysis, the red cells become coated with IgG molecules, which mark the cells for uptake and destruction by splenic macrophages.3 In "cold" AIHA, IgM molecules fix complement to the surface of red blood cells. Rarely, this can lead to activation of the full complement cascade, resulting in red cell lysis, but more often it is stopped at the C3 stage, leading to C3-coated red cells which are then taken up by hepatic macrophages.4
Suspecting the Diagnosis
In many patients, it is the symptoms and signs of anemia that lead to suspicion of hemolysis. Older patients often present earlier in the course of the disease due to lack of tolerance of anemia, especially if there is a sudden drop in the red blood cell count. Dark, cola-colored urine resulting from the presence of free hemoglobin may be noted by some patients. Patients with rapid-onset hemolysis may note lumbar back pain, and those with cold agglutinins often note symptoms related to agglutination of red cells in the peripheral circulation, such as the development of acrocyanosis in cold weather.5 In rare cases, patients will have abdominal pain when eating cold food due to ischemia related to agglutination of red cells in the viscera. Some patients with cold agglutinins can have an exacerbation of their hemolysis with cold exposure.
Unlike patients with immune thrombocytopenia, those with AIHA may have mild splenomegaly on exam. The presence of enlarged lymph nodes or massive splenomegaly should raise concern about concomitant lymphoma or chronic lymphocytic leukemia.
Making the Diagnosis
The 2 key steps in diagnosis are (1) demonstrating hemolysis and (2) demonstrating the autoimmune component.
Laboratory Evaluation for Hemolysis
Hemolysis is proven by finding evidence of both red cell breakdown and the compensatory increase in red cell production this stimulates (Table 2). The following sections discuss the laboratory tests that are performed to investigate hemolysis.
Lactate Dehydrogenase
When red cells undergo hemolysis, they release their contents, which are mostly comprised of hemoglobin but also include lactate dehydrogenase (LDH), an enzyme found in high concentration in red cells. Most patients with hemolysis will have an elevated LDH level, making this a sensitive test. However, because many other processes, including liver disease and pneumonia, also raise the serum LDH level, this finding is not specific for hemolysis.
Serum Bilirubin
Hemoglobin is salvaged by haptoglobin, and the heme moiety is broken down first to bilirubin and then to urobilinogen, which is excreted in the urine.2 Bilirubin produced from the breakdown of heme is not conjugated, but rather is delivered to the liver, where it is conjugated and excreted into the bile. In hemolysis, the concentration of unconjugated bilirubin (indirect bilirubin) is increased, while in liver disease the level of conjugated bilirubin (direct bilirubin) is increased. However, if the patient has concomitant liver disease with an increased direct bilirubin level, the serum bilirubin test is not reliable.
Serum Haptoglobin
Haptoglobin binds free serum hemoglobin and is taken up by the liver. Haptoglobin usually falls to very low levels in hemolysis. A confounder is that haptoglobin is an acute phase reactant and can rise with systemic disease or inflammation. However, patients with advanced liver disease will have low haptoglobin levels due to lack of synthesis, and up to 2% of the population may congenitally lack haptoglobin.1
Serum Hemoglobin
If the hemolysis is very rapid, the amount of free hemoglobin released will overwhelm the binding capacity of haptoglobin and lead to free hemoglobin in the plasma. This can be crudely quantified by examining the plasma color. Even minute amounts of free hemoglobin will turn the plasma pink. In fulminant hemolysis, the plasma will be cola-colored.
Reticulocyte Count
In most patients with hemolysis, the destruction of red cells is accompanied by an increase in the reticulocyte count. Reticulocytes are red cells that still contain RNA and are a marker of red cells that are approximately 24 hours old or less. Traditionally, reticulocytes were measured manually by staining the blood smear with vital blue and counting the percentage of cells that absorb the stain; this percentage needs to be adjusted for the hematocrit. Usually a percentage above 1.5% is considered indicative of an elevated reticulocyte count. Recently, automated complete blood count machines have taken advantage of the fact that reticulocytes will absorb certain stains; these machines can directly measure the reticulocyte count via flow cytometry, which results in an “absolute” reticulocyte count. The reticulocyte count obtained using this method does not have to be corrected for hematocrit, and levels of approximately 90,000/μL are considered raised. However, the reticulocyte count can also be raised in blood loss or in patients who have other causes of anemia (eg, iron deficiency) under treatment. In addition, as many as 25% of patients with AIHA will never have raised counts for various reasons, such as nutritional deficiency, autoimmune destruction of red cell precursors, or lack of erythropoietin.
Blood Smear
The blood smear provides vital information. The hallmark laboratory parameter of AIHA is spherocytes seen on the blood smear. In AIHA, antibodies and/or complement attach to the red cells, and when the antibodies or complement are taken up by macrophages in the spleen some of the red blood cell mem-brane is removed as well, decreasing the surface area of the cell. As the surface area of the red cell decreases with each pass through the spleen, the cell's shape changes from a biconcave disk to a sphere before the cell is destroyed, reflecting the fact that a sphere has the smallest surface area for a given volume. The vast majority of patients with AIHA will have spherocytes on the blood smear. However, spherocytes are not specific to AIHA, as they can be seen in hereditary spherocytosis, Wilson’s disease, clostridial sepsis, and severe burns.
Patients with cold agglutinins will often have red cell agglutination on the blood smear. In addition, patients with AIHA will often have a raised mean corpuscular volume (MCV) for 2 reasons. In patients with brisk reticulocytosis, the MCV will be raised due to the large size of the reticulocyte. In patients with cold agglutinin disease, the MCV may be falsely raised due to clumping of the red blood cells.
Urinary Hemosiderin
When hemoglobin is excreted by the kidney, the iron is deposited in the tubules. When the tubule cells are sloughed off, they appear in the urine. The urine can be stained for iron, and a positive result is another sign of hemolysis. Hemosiderinuria is a later sign of hemolysis, as it takes 1 week for iron-laden tubule cells to be excreted in sufficient quantities to be detected in the urine.
Urinary Hemoglobin
One other sign of hemolysis is the presence of hemoglobin in the urine. A quick way to demonstrate hemoglobinuria is to check the urine with a dipstick followed by a microscopic exam. In hemolysis, the dipstick will detect “blood,” while the microscopic exam will be negative for red cells.
Laboratory Evaluation for Autoimmune Component
The autoimmune component is shown by demonstrating the presence of either IgG molecules or complement on the surface of red blood cells.4,6 This can be done by performing the direct antiglobulin test (DAT) or Coombs test. IgG bound to red cells will not agglutinate them, but if IgM that is directed against IgG or C3 is added, the red cells will agglutinate, proving that there is IgG and/or C3 on the red cell membrane. The finding of a positive DAT in the setting of a hemolytic anemia is diagnostic of AIHA. Beware of individuals with concomitant weak positive DAT and other causes of hemolysis. The strength of the DAT result and the degree of hemolysis must match in order to conclude that the hemolysis is immune-mediated.
There are several pitfalls to the DAT. One is that a positive DAT is found in 1:1000 patients in the normal population and in up to several percent of ill patients, especially those with elevated gamma globulin, such as patients with liver disease or HIV infection.6 Administration of intravenous immunoglobulin (IVIG) can also create a positive DAT. Conversely, patients can have AIHA with a negative DAT.7-9 For some patients, the number of IgG molecules bound to the red cell is below the detection limit of the DAT reagents. Other patients can have IgA or “warm” IgM as the cause of the AIHA.10 Specialty laboratories can test for these possibilities. The diagnosis of DAT-negative AIHA should be made with caution, and other causes of hemolysis, such as hereditary spherocytosis or paroxysmal hematuria, should be excluded.
Transfusion Therapy
Performing transfusions can be very difficult in patients with AIHA.2 The presence of the autoantibody can interfere with typing of the blood and almost always interferes with the crossmatch, since this final step consists of mixing the patient’s serum or plasma with donor red cells. In most patients with AIHA, the autoantibodies will react with any donor cells, rendering a negative crossmatch impossible. Without the crossmatch, the concern is that underlying alloantibodies can be missed. Studies indicate that 15% to 32% of patients will have underlying alloantibodies, which can lead to transfusion reactions.2 However, there are 2 considerations that may mitigate these concerns.11,12 First, patients who have never been transfused or pregnant will rarely have alloantibodies. Second, a patient who has been transfused in the remote past may have an anamnestic antibody response but not an immediate hemolytic reaction.
The transfusion service can take several steps to identify alloantibodies. Occasionally, if the autoantibody is weakly reacting when the patient’s serum is tested against a panel of reagent red cells, the alloantibodies can be identified by their stronger reactions as compared with the weakly reactive autoantibody. The most common technique for identifying alloantibodies is the autoadsorption technique.4,13 This involves incu-bating the patient’s red cells with the patient’s serum to adsorb the autoantibody. After a period of incubation, the cells are pelleted and the serum is collected as the supernatant. The adsorbed serum may be incubated with another sample of the patient’s cells for a second adsorption if the initial agglutination reactions of the patient’s serum with the reagent cells were strong. After 1 to 3 adsorptions, the adsorbed serum is tested with a red cell panel in order to check for “leftover” alloantibodies.
When a patient is first suspected of having AIHA, a generous sample of blood should be given to the transfusion service to allow for adequate testing. Many centers will test the blood not only for blood groups ABO and D but also perform full Rh typing plus check for Kidd, Duffy and Kell status.14 Increasingly, this is performed by direct genetic sequencing for the appropriate genotypes. This can allow transfusion of phenotypically matched red blood cells to lessen the risk of alloantibody formation.
One difficult issue is timing of transfusion. Clinicians are often hesitant to transfuse patients with AIHA due to fear of reactions, but in patients with severe anemia, especially elderly patients or those with heart disease, transfusion can be lifesaving. Since in some cases it may take hours to screen for alloantibodies, it is often preferable to transfuse patients with severe anemia and observe carefully for reaction.
1. Liebman HA, Weitz IC. Autoimmune hemolytic anemia. Med Clin North Am. 2017;101:351-359.
2. Barros MM, Blajchman MA, Bordin JO. Warm autoimmune hemolytic anemia: recent progress in understanding the immunobiology and the treatment. Transfus Med Rev. 2010;24:195–210.
3. Seve P, Philippe P, Dufour JF, et al. Autoimmune hemolytic anemia: classification and therapeutic approaches. Expert Rev Hematol. 2008;1:189-204.
4. Gehrs BC, Friedberg RC. Autoimmune hemolytic anemia. Am J Hematol. 2002;69:258-271.
5. Berentsen S. How I manage cold agglutinin disease. Br J Haematol. 2011;153:309-317.
6. Zantek ND, Koepsell SA, Tharp DR Jr, Cohn CS. The direct antiglobulin test: a critical step in the evaluation of hemolysis. Am J Hematol. 2012;87:707-709.
7. Michel M. Classification and therapeutic approaches in autoimmune hemolytic anemia: an update. Expert Rev Hematol. 2011;4:607-618.
8. Garratty G. Immune hemolytic anemia associated with negative routine serology. Semin Hematol. 2005;42:156-164.
9. Sachs UJ, Roder L, Santoso S, Bein G. Does a negative direct antiglobulin test exclude warm autoimmune haemolytic anaemia? A prospective study of 504 cases. Br J Haematol. 2006;132:655-656.
10. Sokol RJ, Booker DJ, Stamps R, et al. IgA red cell autoantibodies and autoimmune hemolysis. Transfusion. 1997;37:175-181.
11. Petz LD. “Least incompatible” units for transfusion in autoimmune hemolytic anemia: should we eliminate this meaningless term? A commentary for clinicians and transfusion medicine professionals. Transfusion. 2003;43:1503-1507.
12. Blackall DP. How do I approach patients with warm-reactive autoantibodies? Transfusion. 2011;51:14-17.
13. Winkelmayer WC, Liu J, Setoguchi S, Choudhry NK. Effectiveness and safety of warfarin initiation in older hemodialysis patients with incident atrial fibrillation. Clin J Am Soc Nephrol. 2011;6:2662-2668.
14. Ness PM. How do I encourage clinicians to transfuse mismatched blood to patients with autoimmune hemolytic anemia in urgent situations? Transfusion. 2006;46:1859-1862.
The autoimmune hemolytic anemias (AIHA) are rare but important hematologic diseases. They can range in severity from mildly symptomatic illness to a rapidly fatal syndrome. The incidence of AIHA is estimated to be between 0.6 and 3 cases per 100,000 persons.1,2 AIHA is mediated by antibodies, and in the majority of cases immunoglobulin (Ig) G is the mediating antibody. This type of AIHA is referred to as "warm" AIHA because IgG antibodies bind best at body temperature. "Cold" AIHA is mediated by IgM antibodies, which bind maximally at temperatures below 37°C (Table 1). This article series reviews the most common types of AIHA, with an overview of evaluation and diagnosis presented in this article and management of warm, cold, and drug-induced AIHA reviewed in a separate article.
Pathogenesis
In most cases, the ultimate etiology of AIHA is unknown. In warm AIHA, the target epitopes in most cases are Rh proteins.2 What leads the immune system to target these proteins is unidentified, but one theory is that an initial immune response to a foreign antigen starts to cross-react with the Rh proteins and the immune system fails to suppress this autoreactive response, leading to hemolysis. In IgG-mediated (warm) hemolysis, the red cells become coated with IgG molecules, which mark the cells for uptake and destruction by splenic macrophages.3 In "cold" AIHA, IgM molecules fix complement to the surface of red blood cells. Rarely, this can lead to activation of the full complement cascade, resulting in red cell lysis, but more often it is stopped at the C3 stage, leading to C3-coated red cells which are then taken up by hepatic macrophages.4
Suspecting the Diagnosis
In many patients, it is the symptoms and signs of anemia that lead to suspicion of hemolysis. Older patients often present earlier in the course of the disease due to lack of tolerance of anemia, especially if there is a sudden drop in the red blood cell count. Dark, cola-colored urine resulting from the presence of free hemoglobin may be noted by some patients. Patients with rapid-onset hemolysis may note lumbar back pain, and those with cold agglutinins often note symptoms related to agglutination of red cells in the peripheral circulation, such as the development of acrocyanosis in cold weather.5 In rare cases, patients will have abdominal pain when eating cold food due to ischemia related to agglutination of red cells in the viscera. Some patients with cold agglutinins can have an exacerbation of their hemolysis with cold exposure.
Unlike patients with immune thrombocytopenia, those with AIHA may have mild splenomegaly on exam. The presence of enlarged lymph nodes or massive splenomegaly should raise concern about concomitant lymphoma or chronic lymphocytic leukemia.
Making the Diagnosis
The 2 key steps in diagnosis are (1) demonstrating hemolysis and (2) demonstrating the autoimmune component.
Laboratory Evaluation for Hemolysis
Hemolysis is proven by finding evidence of both red cell breakdown and the compensatory increase in red cell production this stimulates (Table 2). The following sections discuss the laboratory tests that are performed to investigate hemolysis.
Lactate Dehydrogenase
When red cells undergo hemolysis, they release their contents, which are mostly comprised of hemoglobin but also include lactate dehydrogenase (LDH), an enzyme found in high concentration in red cells. Most patients with hemolysis will have an elevated LDH level, making this a sensitive test. However, because many other processes, including liver disease and pneumonia, also raise the serum LDH level, this finding is not specific for hemolysis.
Serum Bilirubin
Hemoglobin is salvaged by haptoglobin, and the heme moiety is broken down first to bilirubin and then to urobilinogen, which is excreted in the urine.2 Bilirubin produced from the breakdown of heme is not conjugated, but rather is delivered to the liver, where it is conjugated and excreted into the bile. In hemolysis, the concentration of unconjugated bilirubin (indirect bilirubin) is increased, while in liver disease the level of conjugated bilirubin (direct bilirubin) is increased. However, if the patient has concomitant liver disease with an increased direct bilirubin level, the serum bilirubin test is not reliable.
Serum Haptoglobin
Haptoglobin binds free serum hemoglobin and is taken up by the liver. Haptoglobin usually falls to very low levels in hemolysis. A confounder is that haptoglobin is an acute phase reactant and can rise with systemic disease or inflammation. However, patients with advanced liver disease will have low haptoglobin levels due to lack of synthesis, and up to 2% of the population may congenitally lack haptoglobin.1
Serum Hemoglobin
If the hemolysis is very rapid, the amount of free hemoglobin released will overwhelm the binding capacity of haptoglobin and lead to free hemoglobin in the plasma. This can be crudely quantified by examining the plasma color. Even minute amounts of free hemoglobin will turn the plasma pink. In fulminant hemolysis, the plasma will be cola-colored.
Reticulocyte Count
In most patients with hemolysis, the destruction of red cells is accompanied by an increase in the reticulocyte count. Reticulocytes are red cells that still contain RNA and are a marker of red cells that are approximately 24 hours old or less. Traditionally, reticulocytes were measured manually by staining the blood smear with vital blue and counting the percentage of cells that absorb the stain; this percentage needs to be adjusted for the hematocrit. Usually a percentage above 1.5% is considered indicative of an elevated reticulocyte count. Recently, automated complete blood count machines have taken advantage of the fact that reticulocytes will absorb certain stains; these machines can directly measure the reticulocyte count via flow cytometry, which results in an “absolute” reticulocyte count. The reticulocyte count obtained using this method does not have to be corrected for hematocrit, and levels of approximately 90,000/μL are considered raised. However, the reticulocyte count can also be raised in blood loss or in patients who have other causes of anemia (eg, iron deficiency) under treatment. In addition, as many as 25% of patients with AIHA will never have raised counts for various reasons, such as nutritional deficiency, autoimmune destruction of red cell precursors, or lack of erythropoietin.
Blood Smear
The blood smear provides vital information. The hallmark laboratory parameter of AIHA is spherocytes seen on the blood smear. In AIHA, antibodies and/or complement attach to the red cells, and when the antibodies or complement are taken up by macrophages in the spleen some of the red blood cell mem-brane is removed as well, decreasing the surface area of the cell. As the surface area of the red cell decreases with each pass through the spleen, the cell's shape changes from a biconcave disk to a sphere before the cell is destroyed, reflecting the fact that a sphere has the smallest surface area for a given volume. The vast majority of patients with AIHA will have spherocytes on the blood smear. However, spherocytes are not specific to AIHA, as they can be seen in hereditary spherocytosis, Wilson’s disease, clostridial sepsis, and severe burns.
Patients with cold agglutinins will often have red cell agglutination on the blood smear. In addition, patients with AIHA will often have a raised mean corpuscular volume (MCV) for 2 reasons. In patients with brisk reticulocytosis, the MCV will be raised due to the large size of the reticulocyte. In patients with cold agglutinin disease, the MCV may be falsely raised due to clumping of the red blood cells.
Urinary Hemosiderin
When hemoglobin is excreted by the kidney, the iron is deposited in the tubules. When the tubule cells are sloughed off, they appear in the urine. The urine can be stained for iron, and a positive result is another sign of hemolysis. Hemosiderinuria is a later sign of hemolysis, as it takes 1 week for iron-laden tubule cells to be excreted in sufficient quantities to be detected in the urine.
Urinary Hemoglobin
One other sign of hemolysis is the presence of hemoglobin in the urine. A quick way to demonstrate hemoglobinuria is to check the urine with a dipstick followed by a microscopic exam. In hemolysis, the dipstick will detect “blood,” while the microscopic exam will be negative for red cells.
Laboratory Evaluation for Autoimmune Component
The autoimmune component is shown by demonstrating the presence of either IgG molecules or complement on the surface of red blood cells.4,6 This can be done by performing the direct antiglobulin test (DAT) or Coombs test. IgG bound to red cells will not agglutinate them, but if IgM that is directed against IgG or C3 is added, the red cells will agglutinate, proving that there is IgG and/or C3 on the red cell membrane. The finding of a positive DAT in the setting of a hemolytic anemia is diagnostic of AIHA. Beware of individuals with concomitant weak positive DAT and other causes of hemolysis. The strength of the DAT result and the degree of hemolysis must match in order to conclude that the hemolysis is immune-mediated.
There are several pitfalls to the DAT. One is that a positive DAT is found in 1:1000 patients in the normal population and in up to several percent of ill patients, especially those with elevated gamma globulin, such as patients with liver disease or HIV infection.6 Administration of intravenous immunoglobulin (IVIG) can also create a positive DAT. Conversely, patients can have AIHA with a negative DAT.7-9 For some patients, the number of IgG molecules bound to the red cell is below the detection limit of the DAT reagents. Other patients can have IgA or “warm” IgM as the cause of the AIHA.10 Specialty laboratories can test for these possibilities. The diagnosis of DAT-negative AIHA should be made with caution, and other causes of hemolysis, such as hereditary spherocytosis or paroxysmal hematuria, should be excluded.
Transfusion Therapy
Performing transfusions can be very difficult in patients with AIHA.2 The presence of the autoantibody can interfere with typing of the blood and almost always interferes with the crossmatch, since this final step consists of mixing the patient’s serum or plasma with donor red cells. In most patients with AIHA, the autoantibodies will react with any donor cells, rendering a negative crossmatch impossible. Without the crossmatch, the concern is that underlying alloantibodies can be missed. Studies indicate that 15% to 32% of patients will have underlying alloantibodies, which can lead to transfusion reactions.2 However, there are 2 considerations that may mitigate these concerns.11,12 First, patients who have never been transfused or pregnant will rarely have alloantibodies. Second, a patient who has been transfused in the remote past may have an anamnestic antibody response but not an immediate hemolytic reaction.
The transfusion service can take several steps to identify alloantibodies. Occasionally, if the autoantibody is weakly reacting when the patient’s serum is tested against a panel of reagent red cells, the alloantibodies can be identified by their stronger reactions as compared with the weakly reactive autoantibody. The most common technique for identifying alloantibodies is the autoadsorption technique.4,13 This involves incu-bating the patient’s red cells with the patient’s serum to adsorb the autoantibody. After a period of incubation, the cells are pelleted and the serum is collected as the supernatant. The adsorbed serum may be incubated with another sample of the patient’s cells for a second adsorption if the initial agglutination reactions of the patient’s serum with the reagent cells were strong. After 1 to 3 adsorptions, the adsorbed serum is tested with a red cell panel in order to check for “leftover” alloantibodies.
When a patient is first suspected of having AIHA, a generous sample of blood should be given to the transfusion service to allow for adequate testing. Many centers will test the blood not only for blood groups ABO and D but also perform full Rh typing plus check for Kidd, Duffy and Kell status.14 Increasingly, this is performed by direct genetic sequencing for the appropriate genotypes. This can allow transfusion of phenotypically matched red blood cells to lessen the risk of alloantibody formation.
One difficult issue is timing of transfusion. Clinicians are often hesitant to transfuse patients with AIHA due to fear of reactions, but in patients with severe anemia, especially elderly patients or those with heart disease, transfusion can be lifesaving. Since in some cases it may take hours to screen for alloantibodies, it is often preferable to transfuse patients with severe anemia and observe carefully for reaction.
The autoimmune hemolytic anemias (AIHA) are rare but important hematologic diseases. They can range in severity from mildly symptomatic illness to a rapidly fatal syndrome. The incidence of AIHA is estimated to be between 0.6 and 3 cases per 100,000 persons.1,2 AIHA is mediated by antibodies, and in the majority of cases immunoglobulin (Ig) G is the mediating antibody. This type of AIHA is referred to as "warm" AIHA because IgG antibodies bind best at body temperature. "Cold" AIHA is mediated by IgM antibodies, which bind maximally at temperatures below 37°C (Table 1). This article series reviews the most common types of AIHA, with an overview of evaluation and diagnosis presented in this article and management of warm, cold, and drug-induced AIHA reviewed in a separate article.
Pathogenesis
In most cases, the ultimate etiology of AIHA is unknown. In warm AIHA, the target epitopes in most cases are Rh proteins.2 What leads the immune system to target these proteins is unidentified, but one theory is that an initial immune response to a foreign antigen starts to cross-react with the Rh proteins and the immune system fails to suppress this autoreactive response, leading to hemolysis. In IgG-mediated (warm) hemolysis, the red cells become coated with IgG molecules, which mark the cells for uptake and destruction by splenic macrophages.3 In "cold" AIHA, IgM molecules fix complement to the surface of red blood cells. Rarely, this can lead to activation of the full complement cascade, resulting in red cell lysis, but more often it is stopped at the C3 stage, leading to C3-coated red cells which are then taken up by hepatic macrophages.4
Suspecting the Diagnosis
In many patients, it is the symptoms and signs of anemia that lead to suspicion of hemolysis. Older patients often present earlier in the course of the disease due to lack of tolerance of anemia, especially if there is a sudden drop in the red blood cell count. Dark, cola-colored urine resulting from the presence of free hemoglobin may be noted by some patients. Patients with rapid-onset hemolysis may note lumbar back pain, and those with cold agglutinins often note symptoms related to agglutination of red cells in the peripheral circulation, such as the development of acrocyanosis in cold weather.5 In rare cases, patients will have abdominal pain when eating cold food due to ischemia related to agglutination of red cells in the viscera. Some patients with cold agglutinins can have an exacerbation of their hemolysis with cold exposure.
Unlike patients with immune thrombocytopenia, those with AIHA may have mild splenomegaly on exam. The presence of enlarged lymph nodes or massive splenomegaly should raise concern about concomitant lymphoma or chronic lymphocytic leukemia.
Making the Diagnosis
The 2 key steps in diagnosis are (1) demonstrating hemolysis and (2) demonstrating the autoimmune component.
Laboratory Evaluation for Hemolysis
Hemolysis is proven by finding evidence of both red cell breakdown and the compensatory increase in red cell production this stimulates (Table 2). The following sections discuss the laboratory tests that are performed to investigate hemolysis.
Lactate Dehydrogenase
When red cells undergo hemolysis, they release their contents, which are mostly comprised of hemoglobin but also include lactate dehydrogenase (LDH), an enzyme found in high concentration in red cells. Most patients with hemolysis will have an elevated LDH level, making this a sensitive test. However, because many other processes, including liver disease and pneumonia, also raise the serum LDH level, this finding is not specific for hemolysis.
Serum Bilirubin
Hemoglobin is salvaged by haptoglobin, and the heme moiety is broken down first to bilirubin and then to urobilinogen, which is excreted in the urine.2 Bilirubin produced from the breakdown of heme is not conjugated, but rather is delivered to the liver, where it is conjugated and excreted into the bile. In hemolysis, the concentration of unconjugated bilirubin (indirect bilirubin) is increased, while in liver disease the level of conjugated bilirubin (direct bilirubin) is increased. However, if the patient has concomitant liver disease with an increased direct bilirubin level, the serum bilirubin test is not reliable.
Serum Haptoglobin
Haptoglobin binds free serum hemoglobin and is taken up by the liver. Haptoglobin usually falls to very low levels in hemolysis. A confounder is that haptoglobin is an acute phase reactant and can rise with systemic disease or inflammation. However, patients with advanced liver disease will have low haptoglobin levels due to lack of synthesis, and up to 2% of the population may congenitally lack haptoglobin.1
Serum Hemoglobin
If the hemolysis is very rapid, the amount of free hemoglobin released will overwhelm the binding capacity of haptoglobin and lead to free hemoglobin in the plasma. This can be crudely quantified by examining the plasma color. Even minute amounts of free hemoglobin will turn the plasma pink. In fulminant hemolysis, the plasma will be cola-colored.
Reticulocyte Count
In most patients with hemolysis, the destruction of red cells is accompanied by an increase in the reticulocyte count. Reticulocytes are red cells that still contain RNA and are a marker of red cells that are approximately 24 hours old or less. Traditionally, reticulocytes were measured manually by staining the blood smear with vital blue and counting the percentage of cells that absorb the stain; this percentage needs to be adjusted for the hematocrit. Usually a percentage above 1.5% is considered indicative of an elevated reticulocyte count. Recently, automated complete blood count machines have taken advantage of the fact that reticulocytes will absorb certain stains; these machines can directly measure the reticulocyte count via flow cytometry, which results in an “absolute” reticulocyte count. The reticulocyte count obtained using this method does not have to be corrected for hematocrit, and levels of approximately 90,000/μL are considered raised. However, the reticulocyte count can also be raised in blood loss or in patients who have other causes of anemia (eg, iron deficiency) under treatment. In addition, as many as 25% of patients with AIHA will never have raised counts for various reasons, such as nutritional deficiency, autoimmune destruction of red cell precursors, or lack of erythropoietin.
Blood Smear
The blood smear provides vital information. The hallmark laboratory parameter of AIHA is spherocytes seen on the blood smear. In AIHA, antibodies and/or complement attach to the red cells, and when the antibodies or complement are taken up by macrophages in the spleen some of the red blood cell mem-brane is removed as well, decreasing the surface area of the cell. As the surface area of the red cell decreases with each pass through the spleen, the cell's shape changes from a biconcave disk to a sphere before the cell is destroyed, reflecting the fact that a sphere has the smallest surface area for a given volume. The vast majority of patients with AIHA will have spherocytes on the blood smear. However, spherocytes are not specific to AIHA, as they can be seen in hereditary spherocytosis, Wilson’s disease, clostridial sepsis, and severe burns.
Patients with cold agglutinins will often have red cell agglutination on the blood smear. In addition, patients with AIHA will often have a raised mean corpuscular volume (MCV) for 2 reasons. In patients with brisk reticulocytosis, the MCV will be raised due to the large size of the reticulocyte. In patients with cold agglutinin disease, the MCV may be falsely raised due to clumping of the red blood cells.
Urinary Hemosiderin
When hemoglobin is excreted by the kidney, the iron is deposited in the tubules. When the tubule cells are sloughed off, they appear in the urine. The urine can be stained for iron, and a positive result is another sign of hemolysis. Hemosiderinuria is a later sign of hemolysis, as it takes 1 week for iron-laden tubule cells to be excreted in sufficient quantities to be detected in the urine.
Urinary Hemoglobin
One other sign of hemolysis is the presence of hemoglobin in the urine. A quick way to demonstrate hemoglobinuria is to check the urine with a dipstick followed by a microscopic exam. In hemolysis, the dipstick will detect “blood,” while the microscopic exam will be negative for red cells.
Laboratory Evaluation for Autoimmune Component
The autoimmune component is shown by demonstrating the presence of either IgG molecules or complement on the surface of red blood cells.4,6 This can be done by performing the direct antiglobulin test (DAT) or Coombs test. IgG bound to red cells will not agglutinate them, but if IgM that is directed against IgG or C3 is added, the red cells will agglutinate, proving that there is IgG and/or C3 on the red cell membrane. The finding of a positive DAT in the setting of a hemolytic anemia is diagnostic of AIHA. Beware of individuals with concomitant weak positive DAT and other causes of hemolysis. The strength of the DAT result and the degree of hemolysis must match in order to conclude that the hemolysis is immune-mediated.
There are several pitfalls to the DAT. One is that a positive DAT is found in 1:1000 patients in the normal population and in up to several percent of ill patients, especially those with elevated gamma globulin, such as patients with liver disease or HIV infection.6 Administration of intravenous immunoglobulin (IVIG) can also create a positive DAT. Conversely, patients can have AIHA with a negative DAT.7-9 For some patients, the number of IgG molecules bound to the red cell is below the detection limit of the DAT reagents. Other patients can have IgA or “warm” IgM as the cause of the AIHA.10 Specialty laboratories can test for these possibilities. The diagnosis of DAT-negative AIHA should be made with caution, and other causes of hemolysis, such as hereditary spherocytosis or paroxysmal hematuria, should be excluded.
Transfusion Therapy
Performing transfusions can be very difficult in patients with AIHA.2 The presence of the autoantibody can interfere with typing of the blood and almost always interferes with the crossmatch, since this final step consists of mixing the patient’s serum or plasma with donor red cells. In most patients with AIHA, the autoantibodies will react with any donor cells, rendering a negative crossmatch impossible. Without the crossmatch, the concern is that underlying alloantibodies can be missed. Studies indicate that 15% to 32% of patients will have underlying alloantibodies, which can lead to transfusion reactions.2 However, there are 2 considerations that may mitigate these concerns.11,12 First, patients who have never been transfused or pregnant will rarely have alloantibodies. Second, a patient who has been transfused in the remote past may have an anamnestic antibody response but not an immediate hemolytic reaction.
The transfusion service can take several steps to identify alloantibodies. Occasionally, if the autoantibody is weakly reacting when the patient’s serum is tested against a panel of reagent red cells, the alloantibodies can be identified by their stronger reactions as compared with the weakly reactive autoantibody. The most common technique for identifying alloantibodies is the autoadsorption technique.4,13 This involves incu-bating the patient’s red cells with the patient’s serum to adsorb the autoantibody. After a period of incubation, the cells are pelleted and the serum is collected as the supernatant. The adsorbed serum may be incubated with another sample of the patient’s cells for a second adsorption if the initial agglutination reactions of the patient’s serum with the reagent cells were strong. After 1 to 3 adsorptions, the adsorbed serum is tested with a red cell panel in order to check for “leftover” alloantibodies.
When a patient is first suspected of having AIHA, a generous sample of blood should be given to the transfusion service to allow for adequate testing. Many centers will test the blood not only for blood groups ABO and D but also perform full Rh typing plus check for Kidd, Duffy and Kell status.14 Increasingly, this is performed by direct genetic sequencing for the appropriate genotypes. This can allow transfusion of phenotypically matched red blood cells to lessen the risk of alloantibody formation.
One difficult issue is timing of transfusion. Clinicians are often hesitant to transfuse patients with AIHA due to fear of reactions, but in patients with severe anemia, especially elderly patients or those with heart disease, transfusion can be lifesaving. Since in some cases it may take hours to screen for alloantibodies, it is often preferable to transfuse patients with severe anemia and observe carefully for reaction.
1. Liebman HA, Weitz IC. Autoimmune hemolytic anemia. Med Clin North Am. 2017;101:351-359.
2. Barros MM, Blajchman MA, Bordin JO. Warm autoimmune hemolytic anemia: recent progress in understanding the immunobiology and the treatment. Transfus Med Rev. 2010;24:195–210.
3. Seve P, Philippe P, Dufour JF, et al. Autoimmune hemolytic anemia: classification and therapeutic approaches. Expert Rev Hematol. 2008;1:189-204.
4. Gehrs BC, Friedberg RC. Autoimmune hemolytic anemia. Am J Hematol. 2002;69:258-271.
5. Berentsen S. How I manage cold agglutinin disease. Br J Haematol. 2011;153:309-317.
6. Zantek ND, Koepsell SA, Tharp DR Jr, Cohn CS. The direct antiglobulin test: a critical step in the evaluation of hemolysis. Am J Hematol. 2012;87:707-709.
7. Michel M. Classification and therapeutic approaches in autoimmune hemolytic anemia: an update. Expert Rev Hematol. 2011;4:607-618.
8. Garratty G. Immune hemolytic anemia associated with negative routine serology. Semin Hematol. 2005;42:156-164.
9. Sachs UJ, Roder L, Santoso S, Bein G. Does a negative direct antiglobulin test exclude warm autoimmune haemolytic anaemia? A prospective study of 504 cases. Br J Haematol. 2006;132:655-656.
10. Sokol RJ, Booker DJ, Stamps R, et al. IgA red cell autoantibodies and autoimmune hemolysis. Transfusion. 1997;37:175-181.
11. Petz LD. “Least incompatible” units for transfusion in autoimmune hemolytic anemia: should we eliminate this meaningless term? A commentary for clinicians and transfusion medicine professionals. Transfusion. 2003;43:1503-1507.
12. Blackall DP. How do I approach patients with warm-reactive autoantibodies? Transfusion. 2011;51:14-17.
13. Winkelmayer WC, Liu J, Setoguchi S, Choudhry NK. Effectiveness and safety of warfarin initiation in older hemodialysis patients with incident atrial fibrillation. Clin J Am Soc Nephrol. 2011;6:2662-2668.
14. Ness PM. How do I encourage clinicians to transfuse mismatched blood to patients with autoimmune hemolytic anemia in urgent situations? Transfusion. 2006;46:1859-1862.
1. Liebman HA, Weitz IC. Autoimmune hemolytic anemia. Med Clin North Am. 2017;101:351-359.
2. Barros MM, Blajchman MA, Bordin JO. Warm autoimmune hemolytic anemia: recent progress in understanding the immunobiology and the treatment. Transfus Med Rev. 2010;24:195–210.
3. Seve P, Philippe P, Dufour JF, et al. Autoimmune hemolytic anemia: classification and therapeutic approaches. Expert Rev Hematol. 2008;1:189-204.
4. Gehrs BC, Friedberg RC. Autoimmune hemolytic anemia. Am J Hematol. 2002;69:258-271.
5. Berentsen S. How I manage cold agglutinin disease. Br J Haematol. 2011;153:309-317.
6. Zantek ND, Koepsell SA, Tharp DR Jr, Cohn CS. The direct antiglobulin test: a critical step in the evaluation of hemolysis. Am J Hematol. 2012;87:707-709.
7. Michel M. Classification and therapeutic approaches in autoimmune hemolytic anemia: an update. Expert Rev Hematol. 2011;4:607-618.
8. Garratty G. Immune hemolytic anemia associated with negative routine serology. Semin Hematol. 2005;42:156-164.
9. Sachs UJ, Roder L, Santoso S, Bein G. Does a negative direct antiglobulin test exclude warm autoimmune haemolytic anaemia? A prospective study of 504 cases. Br J Haematol. 2006;132:655-656.
10. Sokol RJ, Booker DJ, Stamps R, et al. IgA red cell autoantibodies and autoimmune hemolysis. Transfusion. 1997;37:175-181.
11. Petz LD. “Least incompatible” units for transfusion in autoimmune hemolytic anemia: should we eliminate this meaningless term? A commentary for clinicians and transfusion medicine professionals. Transfusion. 2003;43:1503-1507.
12. Blackall DP. How do I approach patients with warm-reactive autoantibodies? Transfusion. 2011;51:14-17.
13. Winkelmayer WC, Liu J, Setoguchi S, Choudhry NK. Effectiveness and safety of warfarin initiation in older hemodialysis patients with incident atrial fibrillation. Clin J Am Soc Nephrol. 2011;6:2662-2668.
14. Ness PM. How do I encourage clinicians to transfuse mismatched blood to patients with autoimmune hemolytic anemia in urgent situations? Transfusion. 2006;46:1859-1862.