Paroxysmal Cold Hemoglobinuria Workup

The workup for hemolytic anemia, which will differentiate paroxysmal cold hemoglobinuria from other hemolytic disorders (see DDx), is illustrated in the image below. The Donath-Landsteiner antibody test, direct antiglobulin test (DAT), and 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. For definite diagnosis of a cold antibody autoimmune hemolytic anemia, the cold agglutinin titer should be markedly elevated (> 1:512). Titers greater than 1:64 are likely due to a cold hemagglutinin disease (CHD) antibody, although hemolysis rarely occurs at titers less than 1000. ^[38]

Test for the presence of CD55 or CD59 on the red blood cell (RBC) membrane if paroxysmal nocturnal hemoglobinuria is suspected (see DDx). Flow cytometry is a more sensitive tool to help exclude the presence of paroxysmal nocturnal hemoglobinuria, compared with the classic standard Ham test or sugar water test.

The diagnosis of autoimmune hemolytic anemia (AIHA) is usually straightforward and made on the basis of the following laboratory findings:

• Normocytic or macrocytic anemia
• Normal platelet count
• Reticulocytosis
• Low serum haptoglobin levels
• Elevated lactate dehydrogenase (LDH) level
• Increased indirect bilirubin level
• A positive direct antiglobulin test with a broad-spectrum antibody against immunoglobulin and complement

Two pieces of information are of utmost importance for the clinician to make an appropriate treatment decision:

1. What type of the antibody is involved? 
2. Is the AIHA primary or secondary?

The type of antibody can be identified with the use of monospecific antibodies to immunoglobulin G (IgG) and complement 3d (C3d). When the red cells are coated with IgG or IgG plus C3d, the antibody is usually a warm antibody (warm antibody AIHA [WAIHA]). When the red cells are coated with C3d only, the antibody is often but not always a cold antibody. In some cases (direct antiglobulin test negativity, IgM warm antibodies, cold antibodies with low titers, or Donath-Landsteiner [D-L] antibodies), diagnosis may be difficult, and the expertise of an immune-hematologic laboratory is required.

For the diagnosis of secondary AIHA, a careful history that includes information on the following is helpful:

• Onset (acute or insidious)
• Recent infections
• Recent transfusions
• Exposure to drugs or vaccination
• Signs and symptoms of immune disease (eg, arthritis)
• General clinical condition

The exclusion of a drug-induced hemolytic anemia is particularly important, because stopping the drug is the most effective therapeutic measure in this situation. A clinical examination (to rule out lymphadenopathy and splenomegaly) is needed. ^[39]

Blood cell studies

Findings on complete blood cell (CBC) count, differential, reticulocyte count, absolute reticulocyte count, platelet count, and peripheral blood smear are as follows:

• 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 due 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 reveal poikilocytosis, spherocytes, polychromasia, and nucleated RBCs. Aggregation of the RBCs can occur, but this is considered mild compared with cold hemagglutinin disease. ^[7]

• 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 raise suspicion for this entity. ^{[37, 38, 40]}

• A constellation of peripheral smear findings with high specificity but low sensitivity for paroxysmal cold hemoglobinuria is neutrophil erythrocyte rosettes, erythrocyte couplets without overt agglutination, and active neutrophil erythrophagocytosis. ^[41]

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 (reported as marked heme positivity but only few RBCs).

Hemosiderin associated with a chronic hemolytic process is detectable. Kidney tubular epithelial cells, containing a deposition of hemosiderin, are shed and collected in urine.

Serum chemistry and complement studies

Test results for acute hemolysis are usually positive and include the following:

• Elevated LDH level
• Increased indirect or unconjugated bilirubin levels (particularly prominent if concomitant liver dysfunction is present)
• Low haptoglobin values
• The presence of free plasma hemoglobin

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.

Infectious disease testing

Evaluation for suspected underlying infectious diseases, if clinically warranted, may include the following:

• Test plasma for Treponema pallidum antibody
• Obtain serologic evaluation for the following viruses: measles, mumps, influenza, varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, parvovirus B19, Coxsackie A9
• Perform Gram smear and culture for bacteria such as Haemophilus influenzae, Mycoplasma pneumoniae, and Klebsiella
• Review thick and thin smears for malaria

Direct antiglobulin testing

The direct antiglobulin test (direct Coombs test, DAT) should be performed with monoclonal anti-IgG, polyclonal screening antisera and monoclonal C3 antisera. Polyclonal screening antisera are inadequate for this purpose because they have poor sensitivity to complement components.

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. ^[42]

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. ^{[43, 44, 45, 46]} 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 three 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 below). If the D-L antibody is present, samples 1 and 2 should be positive. As negative controls, the three 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.

Test considerations are as follows:

• The serum reacts equally well with normal adult RBCs and fetal RBCs; only in the rare case of patients homozygous for group P system phenotype p (pp RBCs, which lack P antigen) will the RBCs not react. ^[9]

• 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. ^[47]

• 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. ^[34]

• A review of Donath-Landsteiner testing practices in Canada concluded that in the rare cases of possible paroxysmal cold hemoglobinuria in adults, establishing the diagnosis can be challenging. The review found poor agreement among experts on the interpretation of a positive Donath-Landsteiner test in adults. ^[48]

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.

Although results from imaging studies do not define the diagnosis of paroxysmal cold hemoglobinuria, the findings can assist in identifying an underlying contributory condition. Appropriate imaging studies include a chest radiograph to look for pneumonia or CT scans to look for lymphoproliferative disease in suspected appropriate cases.

Because hemosiderin is a known nephrotoxic agent, patients undergoing severe hemolysis should avoid further exposure to renal irritants such as intravenous pyelogram dye. ^[49]

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.

The need for additional investigations must be determined by history, clinical findings, and the type of antibody. Routine workup relevant for treatment decisions may include abdominal examination by CT scan (eg, to search for splenomegaly, abdominal lymphomas) or a bone marrow examination and a search for clonal immunoglobulins (immune fixation), serum protein electrophoresis, and quantitative determination of immunoglobulins in case of cold antibodies.