Approach Considerations
Standard blood studies for the workup of suspected hemolytic anemia include the following:
- Complete blood cell count
- Peripheral blood smear
- Serum lactate dehydrogenase (LDH) study
- Serum haptoglobin
- Indirect bilirubin
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.
Complete Blood Cell Count
This test can detect an anemia, pancytopenia, and infections. Along with the differential count, a CBC count can help diagnose hematologic malignancies and other hematological 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.[25]
Red blood cell indices
These studies are performed when a CBC count 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 count 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 B-12, 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).
Polychromasia. 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 TTP, HUS, or mechanical damage (see the image below).
Schistocytes (thrombotic thrombocytopenic purpura). 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 hepatic function or cholelithiasis.
Other Laboratory Studies
The following specific studies may be indicated:
- Direct antiglobulin test (DAT)
- Urine free hemoglobin test
- Urine hemosiderin test
- Red blood cell survival test
- Cold agglutinin titer
- Glucose-6-phosphate dehydrogenase (G-6-PD) screen
- Sickle cell screen
The DAT result is usually positive in autoimmune hemolytic anemia, but it may occasionally be negative in this disorder. DAT-negative autoimmune hemolytic anemias have been reviewed.[26] From 5-10% of all autoimmune hemolytic anemias are DAT negative. The polybrene test can detect DAT-negative autoimmune hemolytic anemia.[26] 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.[27]
In addition to hemoglobinuria, urine may be dark as a result of myoglobinuria or porphyria. 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 Cr 51 [51 Cr] 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.
With the cold agglutinin titer, anti-I and anti-I (little i) are elevated in cold agglutinin disease. For example, a high titer of anti-I antibody can be found in mycoplasmal infections and a high titer of anti-i antibody can be found in infectious mononucleosis. In addition, an anti-P cold agglutinin may be seen in paroxysmal cold hemoglobinuria.
A G-6-PD 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 G-6-PD). A positive Heinz body preparation can suggest denatured hemoglobin and thus G-6-PD deficiency (see the image below).
Supra vital stain in hemoglobin H disease that reveals Heinz bodies (golf ball appearance). Screening for sickle cell syndrome 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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