Case of secondary CAD suggests flaws of direct Coombs test

False-negatives in direct Coombs testing reported in up to 10% of CAD

Margarida Maia, PhD avatar

by Margarida Maia, PhD |

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A patient sits on a bed while receiving an intravenous infusion.

Cold agglutinin disease (CAD) secondary to a blood cancer was the most likely diagnosis for an elderly woman in Washington, D.C., despite a negative result on a diagnostic test for CAD known as a direct Coombs test, a case report shows.

The test assesses whether red blood cells are covered with antibodies like those that cause CAD.

The woman’s negative test might partly be explained by low numbers of CAD-driving antibodies, called cold agglutinins, and the fact that these antibodies were of the IgA type, which are undetectable by routine tests that look at the most common type of cold agglutinins known as IgM antibodies.

However, an examination of the patient’s blood sample showed red blood cell clumping at cold temperatures, a sign of CAD, and hemolysis after blood transfusion did not recur in subsequent transfusions using warmed blood. Hemolysis refers to red blood cell destruction.

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‘Unique’ hemolytic transfusion reaction induced by secondary CAD

“We present a unique haemolytic transfusion reaction induced by cold agglutinins associated with [IgA-related blood cancer] …, which may facilitate the diagnoses of other cases requiring thorough [clinical-laboratory] correlation,” the researchers wrote.

The case study, “IgA plasma cell myeloma presenting as cold agglutinin-induced haemolytic transfusion reaction,” was published in BMJ Case Reports.

CAD is an autoimmune disease that occurs when self-reactive cold agglutinins (usually of the IgM type) bind to red blood cells at cold temperatures. This causes the cells to clump together and be flagged for destruction by the immune system, resulting in anemia, or fewer-than-needed red blood cells, and CAD symptoms.

When CAD occurs due to an underlying condition, such as a blood cancer, it’s called secondary CAD. In some types of blood cancer, like multiple myeloma, there is an abnormal growth of B-cells, the immune cells responsible for producing antibodies, including cold agglutinins.

While CAD typically involves cold agglutinins of the IgM type, some studies have reported cold agglutinins associated with B-cell cancers producing antibodies of other types, including IgA and IgG.

In the report, researchers in the U.S. described the case of a woman in her 70s in whom CAD secondary to an IgA multiple myeloma was the most likely cause of her hemolytic reaction to a blood transfusion.

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Woman had history of high blood pressure, thyroid disease, uterine cancer

The woman went to the hospital because she was having trouble breathing during exercise and had been feeling tired for six weeks. She had a history of high blood pressure, thyroid disease, and cancer of the uterus. On examination, her eyes looked pale, but her vital signs were normal.

Blood testing revealed low numbers of red blood cells, white blood cells, and platelets, a condition called pancytopenia. It also showed signs of a form of anemia linked to chronic diseases such as cancer, certain infections, and autoimmune and inflammatory diseases.

To treat her anemia, the woman received a blood transfusion, which resulted in an increase in the levels of hemoglobin, the protein in red blood cells that carries oxygen in the body. However, two weeks later, she returned to the hospital with trouble breathing when exercising. Lab work showed her hemoglobin levels had dropped.

After receiving another blood transfusion, the woman reported dark bloody urine and trouble swallowing. Blood examination revealed that the plasma — the colorless liquid part of the blood — had turned red, indicating hemolysis. This was supported by the presence of several other markers of hemolysis.

A routine direct Coombs test, designed to assess whether IgG antibodies and/or other immune proteins involved in CAD are attached to a patient’s red blood cells, came back negative.

However, when the doctors looked at a pre-transfusion sample of her blood under a microscope, they saw that red blood cells were clumping together at cold temperatures, but this disappeared when the sample was warmed. This was suggestive of the presence of cold agglutinins.

Blood samples collected four days after transfusion were sent to reference laboratories to see if cold agglutinins (of IgM, IgG, and IgA types) could be detected, but these also came back negative. This may have been related to the fact that the woman was no longer experiencing hemolysis when her blood sample was collected, the team noted.

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Bone marrow biopsy revealed presence of IgA multiple myeloma

Further blood testing detected high levels of IgA antibodies. A bone marrow biopsy — a procedure in which a small sample of bone is removed — revealed the presence of an IgA multiple myeloma.

Whenever the woman needed a transfusion due to low hemoglobin levels, the blood was warmed before the transfusion. Using this approach, subsequent blood transfusions were “performed uneventfully,” the researchers wrote.

A diagnosis of secondary CAD appeared to be “the best explanation for this clinical presentation,” the researchers wrote.

The IgA multiple myeloma was treated with a combination of the anti-cancer medications cyclophosphamide and bortezomib alongside dexamethasone, a steroid. The cancer entered into complete remission, meaning it disappeared.

CAD “triggered by [an] unwarmed transfusion in association with … myeloma is rare and may represent a diagnostic challenge,” in part due to the possibility of a false-negative direct Coombs test result, the team wrote.

False-negatives in direct Coombs testing have “been reported in up to 10% of CAD due to various factors, including low sensitivity, non-IgG type [cold] agglutinins (undetectable by the routine IgG-based tests used in our hospital), and delayed testing (as was probably the case of the samples we sent to reference laboratories),” the researchers concluded.