Approach Considerations
Standard blood studies for the workup of suspected hemolytic anemia include the following:
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Complete blood cell count (CBC)
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Peripheral blood smear
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Serum lactate dehydrogenase (LDH)
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Serum haptoglobin
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Indirect bilirubin
Hemolysis of collected blood is more likely to occur in standard large vacuum tubes than in small tubes. Thus, it might better to collect blood for these tests in small vacuum tubes, since hemolysis of collected blood can skew the results of these tests. [36]
Changes in the LDH and serum haptoglobin levels are the most sensitive general tests because the indirect bilirubin is not always increased.
Other laboratory studies may be directed by history, physical examination, peripheral smear, and other laboratory findings. Ultrasonography is used to estimate the spleen size, since the physical examination occasionally does not detect significant splenomegaly. Chest radiography, electrocardiography (ECG), and other studies are used to evaluate cardiopulmonary status.
See the algorithm below.
Complete Blood Cell Count
This test can detect an anemia, pancytopenia, and infections. Along with the differential count, a CBC can help diagnose hematologic malignancies and other hematologic disorders. The platelet count usually is normal in most hemolytic anemias.
Thrombocytopenia can occur in systemic lupus erythematosus (SLE), chronic lymphocytic leukemia (CLL), and microangiopathic hemolytic anemias (eg, patients with defective prosthetic cardiac valves, thrombotic thrombocytopenic purpura [TTP], hemolytic uremic syndrome [HUS], and disseminated intravascular coagulation [DIC]). Thrombocytopenia associated with a positive direct Coombs test occurs in the Evans syndrome. [37]
Red blood cell indices
These studies are performed when a CBC is requested. A low mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) are consistent with a microcytic hypochromic anemia due to iron deficiency that may occur in chronic intravascular hemolysis.
A high MCV is consistent with a macrocytic anemia. Folate consumed during chronic hemolysis may lead to megaloblastosis and a high MCV. However, the MCV also may be elevated in patients with high reticulocyte counts since these cells are larger than mature RBCs. A high MCH and mean corpuscular hemoglobin concentration (MCHC) would suggest spherocytosis.
Red blood cell distribution width study
The red blood cell distribution width (RDW) study is usually performed when a CBC is requested. An increased RDW is a measure of anisocytosis that can occur in hemolytic anemias.
Reticulocyte count
An increased reticulocyte count represents increased RBC production and is a criterion for hemolysis but is not specific for hemolysis. In addition to hemolysis, increased reticulocytes may be a response to blood loss or the treatment of iron, vitamin B12, or folate deficiencies. The reticulocyte count may be normal or low in patients with bone marrow suppression despite ongoing severe hemolysis (aplastic crisis).
Peripheral Blood Smear
Peripheral smear findings can help in the diagnosis of a concomitant underlying hematologic malignancy associated with hemolysis. For example, smears in CLL are characterized by an abundance of small lymphocytes and smudge cells (ruptured CLL cells).
The following are examples of the value of evaluating peripheral blood smears.
Polychromasia indicates RBC immaturity (see the image below).
A peripheral smear may demonstrate spherocytes, suggesting congenital spherocytosis or autoimmune hemolytic anemia (AIHA); see the image below.
Spherocytes. One arrow points to a spherocyte; the other, to a normal RBC with central pallor.
The presence of schistocytes (fragmented red blood cells) suggests DIC, TTP, HUS, or mechanical damage (see the image below).
Lactate Dehydrogenase Study
Serum LDH elevation is a criterion for hemolysis.
LDH elevation is sensitive for hemolysis, but is not specific since LDH is ubiquitous and can be released from neoplastic cells, the liver, or from other damaged organs.
Although an increase in LDH isozymes 1 and 2 is more specific for red blood cell destruction, these enzymes are also increased in patients with myocardial infarction.
Serum Haptoglobin
A low serum haptoglobin level is a criterion for moderate-to-severe hemolysis.
A decrease in serum haptoglobin is more likely in intravascular hemolysis than in extravascular hemolysis.
However, it is an acute phase reactant. Therefore, haptoglobin levels can be normal or elevated despite significant hemolysis in patients with infections and in other reactive states.
Indirect Bilirubin
Unconjugated bilirubin is a criterion for hemolysis, but it is not specific because an elevated indirect bilirubin level also occurs in Gilbert disease.
With hemolysis, the level of indirect bilirubin usually is less than 3 mg/dL. Higher levels of indirect bilirubin indicate compromised liver function or cholelithiasis.
Other Laboratory Studies
The following specific studies may be indicated:
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Direct antiglobulin test (DAT)
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Plasma free hemoglobin
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Urine free hemoglobin test
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Urine hemosiderin test
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Red blood cell survival test
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Cold agglutinin titer
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Glucose-6-phosphate dehydrogenase (G6PD) screen
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Sickle cell screen
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Flow cyometry for paroxysmal nocturnal hemoglobinuria (PNH)
The DAT result is usually positive in autoimmune hemolytic anemia (AIHA), but it may occasionally be negative in this disorder. DAT-negative AIHAs have been reviewed. [38] Approximately 5-10% of all AIHAs are DAT negative. The polybrene test can detect DAT-negative AIHA. [38] In addition, the immunoradiometric assay (IRMA) for red blood cell–bound IgG can be used to diagnose AIHA in patients whose autoantibody levels are too low to be detected by conventional DAT. [39] DAT-negative AIHA has a better prognosis than DAT-positive AIHA. [40]
A promising new method for diagnosing AIHA measures the binding of red blood cells that have autoimmune IgG on their surface to films containing a monoclonal antibody against human immunoglobulin. This test could potentially be used in place of the direct Coombs test, which is prone to false positives. [41]
MicroRNA analysis has been found to be helpful in the diagnosis of AIHA in patients with chronic lymphocytic leukemia. [42]
In addition to hemoglobinuria, either myoglobinuria or porphyria may result in dark urine. To rule out these possibilities, the urine should be tested for free hemoglobin. Hemoglobinuria occurs when the amount of free hemoglobin released during hemolysis exceeds available haptoglobin.
Urine hemosiderin may suggest severe or intravascular hemolysis. Hemosiderin is detected in iron-stained urinary sediment in sloughed renal epithelial cells. The source of urinary hemosiderin is hemoglobinuria that occurs in severe and intravascular hemolysis. When urinary hemoglobin is reabsorbed by renal tubular cells, it is processed to hemosiderin. Therefore, urinary hemosiderin reflects hemoglobinuria and suggests severe or intravascular hemolysis.
Red blood cell survival (chromium-51 [51Cr] survival) is rarely used, but it can definitively demonstrate shortened red blood cell survival (hemolysis). If available, this test can be helpful when the clinical history and laboratory studies cannot establish a diagnosis of hemolysis.
The cold agglutinin titer will be elevated in cold agglutinin disease, with the specific IgM antibody varying according to the underlying disorder. [43] For example, a high titer of anti-I antibody (ie, antibody directed against the I antigen found on normal adult RBCs) occurs in mycoplasmal infections. High titers of anti-i antibody (antibody directed against the i antigen found on fetal cord blood RBCs) have been reported in infectious mononucleosis. An anti-P cold agglutinin may be seen in paroxysmal cold hemoglobinuria.
A G6PD screening can usually detect deficiency of this enzyme, but results can be normal if the reticulocyte count is elevated (reticulocytes contain a considerable amount of G6PD). A positive Heinz body preparation on peripheral smear is suggestive of denatured hemoglobin and thus G6PD deficiency (see the image below).
Supravital stain in hemoglobin H disease reveals Heinz bodies (golf ball appearance).
Screening for sickle cell disease is performed by demonstrating sickling under reduced conditions (sickle cell preparations) and testing for hemoglobin solubility (see the image below). Hemoglobin electrophoresis confirms the presence of abnormal hemoglobin.
Peripheral blood smear with sickled cells at 1000X magnification. Image courtesy of Ulrich Woermann, MD.
Other tests may be indicated to diagnose hereditary spherocytosis, hematologic malignancy, and rarer types of hemolytic anemias.
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Polychromasia.
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Spherocytes. One arrow points to a spherocyte; the other, to a normal RBC with central pallor.
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Schistocytes (thrombotic thrombocytopenic purpura).
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Peripheral blood smear with sickled cells at 1000X magnification. Image courtesy of Ulrich Woermann, MD.
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Supravital stain in hemoglobin H disease reveals Heinz bodies (golf ball appearance).
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Hemolytic anemia algorithm.
