Donath-Landsteiner Hemolytic Anemia Workup

The diagnosis of Donath-Landsteiner hemolytic anemia (DLHA) depends on the performance of the specific test to detect the causative autoantibody, the Donath-Landsteiner (D-L) autoantibody. The clinician must have a high index of suspicion. Donath-Landsteiner hemolytic anemia can be confused with cold agglutinin disease and with warm-antibody autoimmune hemolytic anemia.

Anemia may be mild, moderate, or severe. Anemia is usually normocytic and normochromic. However, the mean corpuscular volume (MCV) may be elevated when reticulocytosis is present. Reticulocytosis does not develop until later in the disease; in the early stages, reticulocytopenia is often present.

Leukopenia may be present early, but the leukocyte count then improves to within the reference range or higher. Leukocytosis may be present during hemolytic episodes.

The peripheral blood smear exhibits spherocytosis, polychromasia, nucleated RBCs, anisocytosis, poikilocytosis, and sometimes erythrophagocytosis by neutrophils (see the images below).^{[13, 14]}

Blood typing should be performed on all patients in the event a blood transfusion is needed. Unfortunately, the presence of autoantibodies may interfere with typing. The autoantibody may react with the RBCs of all potential donors, making detection of alloantibodies difficult. Compatibility testing can be improved by performing the test at 37°C.

The direct antiglobulin test (DAT) with anti-IgG usually produces negative results because of dissociation of IgG from the RBC surface at warm temperatures. The DAT may produce weakly positive results if run at a cold temperature. A positive DAT result is due to C3.

The indirect antiglobulin test, if performed, must be carried out at a cold temperature.

Assays using labeled monoclonal anti-immunoglobulin (Ig) G are preferable. The autoantibody is IgG with anti-P specificity, although the specificity may be for other antigens. The titer is usually less than 1:100.

Donath-Landsteiner Bithermic Hemolytic Test

Positive results are demonstrated by incubation in cold, followed by incubation at 37°C in the presence of complement.

This is a hemolytic assay in which the patient's serum is incubated with normal RBCs and complement at 4°C to allow the early components of complement to be fixed. Subsequently, the specimen is incubated at 37°C in order to allow the later components of complement to be activated. The membrane attack complex lyses the RBCs.

The procedure for this test is as follows:

• Incubate normal papainized group O RBCs (papainization exposes RBC membrane P -antigen sites) and normal serum (as complement source) with the patient's serum (containing D-L antibody) at 0-4°C; the use of papainized pooled O-cells results in exposure of more antigen sites on the cell membrane to the antibody.
• Incubate a second group of samples, as above, at 37°C
• Incubate a third group first at 0-4°C for 30 minutes, then at 37°C for 60 minutes

The presence of hemolysis in the third group without hemolysis in either the first or second group of samples constitutes a positive test result and a diagnosis of Donath-Landsteiner hemolytic anemia.

The Donath-Landsteiner test has a low sensitivity, and results are positive only when the serum antibody titer is high and the test is performed precisely.

A false negative test may be seen if the following is present:

• Low level of antibody due to consumption during hemolysis
• Low complement levels from consumption during hemolysis (this can be prevented by mixing the patient sample with fresh normal donor serum as a source of complement)
• If donor serum as a source of complement is added, presence of globoside in the serum may neutralize the Donath-Landsteiner (D-L) antibody^[15]

Autoantibody specificity may be indicated if D-L test result is positive. Almost all cases are associated with anti-P specificity.

Evidence of hemolysis is elevated levels of indirect bilirubin and lactate dehydrogenase in the presence of a decreased haptoglobin level.

Anemia may be mild, moderate, or severe.

Blood urea nitrogen (BUN) and creatinine levels may be elevated if renal insufficiency is present.

Complement levels are typically decreased.

On serologic testing, results for syphilis, mycoplasmal infection, or viruses (eg, influenza A, measles, mumps, adenovirus, cytomegalovirus, varicella, Epstein-Barr virus [EBV]) may be positive, depending on the underlying cause.

Microscopic examination of the peripheral blood may show RBC agglutination and anisopoikilocytosis. Erythrophagocytosis by neutrophils may also be seen.^[13]

Reticulocytosis is expected. If the etiology of the condition is parvovirus B-19 infection, reticulocytopenia may occur.

Hemoglobinuria, methemoglobinuria, and hemosiderinuria are often present but are not required for diagnosis.

Proteinuria may be present.