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Donath-Landsteiner Hemolytic Anemia Workup

  • Author: Trisha Simone Tavares, MD, FAAP; Chief Editor: Max J Coppes, MD, PhD, MBA  more...
 
Updated: Apr 07, 2014
 

Approach Considerations

The diagnosis of Donath-Landsteiner hemolytic anemia (DLHA) can be elusive. The clinician must have a high index of suspicion.

Laboratory testing is crucial to making the diagnosis, and the medical team should be familiar with the assays available in the treating facility's laboratory.

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Complete Blood Cell Count and Peripheral Smear

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 observed.

Leukopenia with or without neutropenia may be present early, but the leukocyte count gradually improves to within the reference range or higher. Leukocytosis may be present during an acute hemolytic episode.

The peripheral blood smear exhibits spherocytosis, polychromasia, nucleated RBCs, anisocytosis, poikilocytosis, and sometimes erythrophagocytosis by neutrophils (see the images below).[17, 19]

Blood smear showing spherocytosis, polychromatophi Blood smear showing spherocytosis, polychromatophilia, and erythrophagocytosis by neutrophils.
Blood smear showing spherocytosis, polychromatophi Blood smear showing spherocytosis, polychromatophilia, and erythrophagocytosis by neutrophils.
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Blood Typing

Blood typing should be performed on all patients even if anemia is mild. This is important because the hemoglobin may fall acutely and transfusion may be needed. Often, the presence of autoantibodies can interfere with blood typing. The D-L autoantibody may react with the RBCs of potential donors, making detection of alloantibodies difficult. Compatibility testing can be improved by performing typing at 37°C.

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Direct Antiglobulin Test

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.

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Indirect Antiglobulin Test

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

This is a hemolytic assay in which the patient's serum is incubated with normal RBCs and complement at 0-4°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 [20]

Autoantibody specificity may be indicated if D-L test result is positive. Most cases are associated with anti-P specificity, but other specificities have been reported.

Testing should also be performed to determine whether the D-L antibody is IgG or IgM class.

If the D-L test is negative and there is a high index of suspicion, the negative result may be false. More sensitive tests exist. Discuss what other assays may be available in the local laboratories.

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Blood Chemistry and Serology

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 abnormal 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.

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Peripheral Blood Smear

Microscopic examination of the peripheral blood may show spherocytosis, RBC agglutination and anisopoikilocytosis. Erythrophagocytosis by neutrophils may also be seen.[17]

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

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Urinalysis

Hemoglobinuria, methemoglobinuria, and hemosiderinuria are often present but are not required for diagnosis (see urinalysis).

Proteinuria may also be noted.

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Contributor Information and Disclosures
Author

Trisha Simone Tavares, MD, FAAP Attending Physician, Department of Pediatrics, Section of Hematology/Oncology, Cardon Children's Medical Center

Trisha Simone Tavares, MD, FAAP is a member of the following medical societies: Children's Oncology Group

Disclosure: Nothing to disclose.

Coauthor(s)

M Monica Gramatges, MD Assistant Professor, Department of Pediatrics, Section of Hematology-Oncology, Baylor College of Medicine

M Monica Gramatges, MD is a member of the following medical societies: American Association for Cancer Research, American Society of Hematology, American Society of Pediatric Hematology/Oncology, Children's Oncology Group

Disclosure: Nothing to disclose.

Chief Editor

Max J Coppes, MD, PhD, MBA Executive Vice President, Chief Medical and Academic Officer, Renown Heath

Max J Coppes, MD, PhD, MBA is a member of the following medical societies: American College of Healthcare Executives, American Society of Pediatric Hematology/Oncology, Society for Pediatric Research

Disclosure: Nothing to disclose.

Acknowledgements

Nicolas A Camilo, MD Consulting Staff, Division of Pediatric Hematology-Oncology, Mountain States Tumor Institute, St Luke's Regional Medical Center

Nicolas A Camilo, MD is a member of the following medical societies: American Society of Pediatric Hematology/Oncology

Disclosure: Nothing to disclose.

Gary D Crouch, MD Program Director of Pediatric Hematology-Oncology Fellowship, Department of Pediatrics, Associate Professor, Uniformed Services University of the Health Sciences

Gary D Crouch, MD is a member of the following medical societies: American Academy of Pediatrics and American Society of Hematology

Disclosure: Nothing to disclose.

Michael R Jeng, MD Associate Professor, Department of Pediatrics, Division of Hematology/Oncology, Stanford University School of Medicine

Michael R Jeng, MD is a member of the following medical societies: American Society of Hematology, American Society of Pediatric Hematology/Oncology, and Histiocyte Society

Disclosure: Nothing to disclose.

Gary R Jones, MD Associate Medical Director, Clinical Development, Berlex Laboratories

Gary R Jones, MD is a member of the following medical societies: American Academy of Pediatrics, American Society of Pediatric Hematology/Oncology, and Western Society for Pediatric Research

Disclosure: Nothing to disclose.

Thomas W Loew, MD Clinical Professor of Pediatrics, Division Director of Pediatric Hematology/Oncology, University of Missouri Children's Hospital

Thomas W Loew, MD is a member of the following medical societies: American Academy of Pediatrics, American Academy of Pediatrics, American College of Physician Executives, American Medical Association, American Society of Clinical Oncology, American Society of Hematology, American Society of Pediatric Hematology/Oncology, and Children's Oncology Group

Disclosure: Genzyme Grant/research funds Independent contractor; Genzyme Honoraria Speaking and teaching; Amicus Grant/research funds Independent contractor; Purdue Pharmaceuticals Grant/research funds Independent contractor

Mary L Windle, PharmD Adjunct Associate Professor, University of Nebraska Medical Center College of Pharmacy; Editor-in-Chief, Medscape Drug Reference

Disclosure: Nothing to disclose.

References
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Blood smear showing spherocytosis, polychromatophilia, and erythrophagocytosis by neutrophils.
Blood smear showing spherocytosis, polychromatophilia, and erythrophagocytosis by neutrophils.
 
 
 
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