eMedicine Specialties > Hematology > Immune System and Disorders

Hemoglobinuria, Paroxysmal Cold: Differential Diagnoses & Workup

Author: Corinne Goldberg, MD, Fellow in Transfusion Medicine/Blood Banking, Transfusion Medicine Department, Hoxworth Blood Center, University of Cincinnati
Coauthor(s): Ronald A Sacher, MB, BCh, MD, FRCPC, Professor, Internal Medicine and Pathology, Director, Hoxworth Blood Center, University of Cincinnati Academic Health Center
Contributor Information and Disclosures

Updated: Nov 21, 2008

Differential Diagnoses

Cold Agglutinin Disease
Drug-Induced Hemolysis
Malaria
Paroxysmal Nocturnal Hemoglobinuria
Transfusion Reactions

Other Problems to Be Considered

• Cold hemagglutinin disease manifests as an immune hemolytic anemia with acrocyanosis and, rarely, hemoglobinuria following cold exposure. In the same manner as paroxysmal cold hemoglobinuria, the cold hemagglutinin disease antibody, usually IgM, binds in colder areas of the body, allowing interaction with complement (ie, C3), followed by dissociation of the antibody upon circulating through warmer anatomic regions. The complement remains attached to the erythrocyte and is degraded into its intermediate by-products (ie, C3b, iC3b). This is a decisive point that deviates from the paroxysmal cold hemoglobinuria pathway, as the cold hemagglutinin disease route results in the recognition and clearance of the complement coated RBC by the reticuloendothelial system. Completion of the complement cascade, resulting in intravascular hemolysis, does not occur. Although laboratory analysis reveals a similar paroxysmal cold hemoglobinuria direct agglutinin test (DAT) positivity that is restricted to the monoclonal anti-C3, the D-L test is negative.²⁶
• Paroxysmal nocturnal hemoglobinuria (PNH) may be confused with paroxysmal cold hemoglobinuria because of its similar presentation of hemoglobinuria, hemoglobinemia, and anemia secondary to intravascular hemolysis. However, paroxysmal nocturnal hemoglobinuria pathogenesis is dependent on a mutated cellular enzyme called phosphatidylinositol glycan A (PIG-A) that is required to anchor proteins known as glycophosphatidylinositols (GPIs) to the cellular membrane. The absence or marked reduction of expression of GPI antigens such as CD59 and CD55, which have inhibitory complement function, can result in unimpeded destruction of various hematopoietic cells including erythrocytes. In addition, paroxysmal nocturnal hemoglobinuria characteristically manifests with persistent cytopenias and absent bone marrow iron.Testing for paroxysmal nocturnal hemoglobinuria depends on the absence of the GPI antigens determined via various tests, including the highly sensitive flow cytometry and the bacterial toxin aerolysin assay.²⁷ DAT results are negative, as are results from the D-L antibody test.^{28,29,30,31,32}
• Hemolytic transfusion reactions can result in life-threatening intravascular hemolysis and hemoglobinuria upon transmission of incompatible blood products. The recipient's preformed alloantibodies interact with the donor's RBCs, activating the complement sequence. Generally, these antibodies are IgG in origin, resulting in both polyclonal and monoclonal anti-IgG DAT positivity. Clinical history is important for identifying this condition and providing immediate therapeutic intervention.
• Malaria caused by Plasmodium falciparum infection is a fulminating disease that may manifest with episodic, massive, intravascular hemolysis and hemoglobinuria, with systemic symptoms of fever and pain. Peripheral blood smears may identify the pathogens and serologic studies can confirm their presence.
• Warm autoimmune hemolytic anemia (ie, direct Coombs-positive or warm antibody–induced hemolytic anemia) is caused by an antibody that binds to RBCs at 37°C, in contrast to the D-L antibody, which binds to RBCs in the cold. The antibody specificity in autoimmune hemolytic anemia is to the Rh system, and hemoglobinuria is uncommon.
• Myoglobinuria may be misinterpreted as hemoglobinuria at preliminary urine analysis. This condition is generally precipitated by exertion or trauma. RBC hemolysis is not a feature of this disease.

Workup

Laboratory Studies

• Complete blood cell (CBC) count, differential, platelet count, and review of the peripheral blood smear
  • A sudden onset of a marked normochromic, normocytic, or macrocytic anemia may be noted in paroxysmal cold hemoglobinuria, particularly in a severe attack.
  • The reticulocyte count is usually low during an acute episode, representing an ineffective marrow response either to viral hematopoietic suppression or preferential destruction of RBCs by the D-L antibody. Reticulocytosis occurs with resolution of the antibody. 
  • Examination of the peripheral blood smear may briefly reveal the presence of poikilocytosis, spherocytes, polychromasia, and nucleated RBCs. Aggregation of the RBCs can occur, but this is considered mild compared with cold hemagglutinin disease.⁶
  • Monocytes and granulocytes may show erythrophagocytosis. Although more commonly seen in other types of autoimmune hemolytic anemia, the specific presence of RBC engulfment by neutrophils within the peripheral blood has a stronger association with paroxysmal cold hemoglobinuria and should make the observer suspicious for this entity.^32,33
• Urinalysis 
  • In the early part of an acute attack, the urine is dark red-brown because of the presence of free hemoglobin or methemoglobin. Hematuria is generally absent in paroxysmal cold hemoglobinuria, although a minimal quantity of RBCs can be seen.
  • Hemosiderin associated with a chronic hemolytic process is detectable.  Kidney tubular epithelial cells, containing a deposition of hemosiderin, are shed and collected in urine.
• Biochemical testing: Test results for acute hemolysis are usually positive and include an elevated lactate dehydrogenase (LDH) level, increased indirect or unconjugated bilirubin levels (particularly prominent if concomitant liver dysfunction is present), low haptoglobin values, and the presence of free plasma hemoglobin.
• Complement levels: Due to consumption during the acute phase of massive hemolysis, measured plasma complement levels, such as C2, C3, and C4, are decreased in paroxysmal cold hemoglobinuria.
• Evaluation of suspected underlying infectious diseases, if clinically warranted:
  • Test plasma for Treponema pallidum antibody.
  • Obtain serologic evaluation for the following viruses: measles, mumps, influenza, VZV, CMV, EBV, adenovirus, parvovirus B19, Coxsackie A9.
  • Perform Gram smear and culture for bacteria such as H influenzae, M pneumoniae, and K.
  • Review thick and thin smears for malaria.
• Direct antiglobulin test (ie, direct Coombs test, DAT)
  • Monoclonal C3 antisera generally show DAT positivity due to C3d fragments on the RBCs. This reaction occurs during or shortly after the acute paroxysmal cold hemoglobinuria hemolytic episode.³⁴
  • Polyclonal screening antisera are inadequate for this purpose because they have poor sensitivity to complement components.
  • Monoclonal anti-IgG DAT results are usually negative. This is thought to be due to the restrictive thermal range of the D-L antibody, which dissociates at the warmer temperatures at which the DAT is generally performed.^35,36,37,38  Alternatively, if the blood is tested at cold temperatures, then the DAT result may also be positive.
• The Donath-Landsteiner test:
  • The procedure involves incubating 3 specimens: (1) the patient's serum, (2) a mix of patient's and normal serum, and (3) normal serum with P-positive RBCs at 4°C. The sample is heated to 37°C, followed by visual analysis of the serum for hemolysis, which is indicative of a positive reaction (see Image 1). If the D-L antibody is present, samples 1 and 2 should be positive. As negative controls, the set of 3 samples are replicated at testing conditions in which temperature is maintained at 4°C and 37°C throughout. 
  • Because complement may be readily consumed during sample processing, leading to a false-negative result, normal ABO-compatible serum is provided as an additional complement source.
  • The serum reacts equally well with normal adult RBCs and fetal RBCs. Only the rare pp RBCs (homozygous for the absence of P antigen) do not react.⁸
  • Modification of the Donath-Landsteiner test is done to enhance the antigenicity of the RBCs. This is performed by exposing the erythrocytes to an enzyme, papain, treatment, which then further unveils group P antigens.³⁹
  • The specificity of the antibody-mediated hemagglutination and antibody-complement–mediated hemolysis can be confirmed further by inhibition of these processes by globoside and Forssman glycosphingolipid.²⁹
• Indirect antiglobulin test
  • Another interesting technique is to demonstrate the D-L antibody with a modified indirect Coombs test. Control (normal) RBCs are incubated with the patient's serum that contains the D-L antibody. These RBCs are washed with ice-cold saline solution to avoid dissociating the D-L antibody from the RBCs. Monoclonal IgG antiserum is then added.
  • This test is a sensitive indicator of the presence of the D-L antibody in the patient's serum. Note that the antibody in cold agglutinin disease is usually an IgM. 
• Conclusions
  • The D-L antibody test, DAT, and the indirect antiglobulin test are all useful in confirming the clinical diagnosis of paroxysmal cold hemoglobinuria.
  • If the Donath-Landsteiner test results are negative or equivocal and a cold reacting antibody is still suspected, perform a cold agglutinin titer. Titers greater than 64 are likely due to a CHD antibody, although hemolysis rarely occurs at titers less than 1000.³³
  • Test for the presence of CD55 or CD59 on the RBC membrane if paroxysmal nocturnal hemoglobinuria is suspected (see Differentials). Flow cytometry is a more sensitive tool to help exclude the presence of paroxysmal nocturnal hemoglobinuria when compared with the classic standard Ham test or sugar water test.

Imaging Studies

Although results from imaging studies do not define the diagnosis of paroxysmal cold hemoglobinuria, the findings can assist in identifying an underlying contributory condition.

Because hemosiderin is a known nephrotoxic agent, patients undergoing severe hemolysis should avoid further exposure to renal irritants such as intravenous pyelogram dye.⁴⁰

Other Tests

The presence of lymphadenopathy is suggestive of infection, lymphoma, or other underlying disease. Excisional biopsy of enlarged lymph nodes, with flow cytometry and gene rearrangement studies, may prove useful in such cases.

• Search for CME/CE on This Topic »

• Paroxysmal Nocturnal Hemoglobinuria (Hematology)
• Anemia (Hematology)
• Donath-Landsteiner Hemolytic Anemia (Pediatrics: General Medicine)
• Hyperbilirubinemia, Unconjugated (Gastroenterology)

• Immunological Complications of Blood Transfusion
• Renal Hemosiderosis
• Budd-Chiari Syndrome