Researchers call for standard antibody tests to diagnose CAD
Different testing methods lead to different outcomes, study finds
Laboratories testing for cold agglutinins — the self-reactive antibodies causing cold agglutinin disease (CAD) — need to use standard protocols to provide accurate results, compare results between studies, and guide treatment, researchers said.
“International standardization of CA [cold agglutinin] testing for diagnostic use is essential,” the researchers wrote.
The study, “Unifying serological testing for cold agglutinins,” was published as a short report in Vox Sanguinis, the International Journal of Transfusion Medicine. It was funded by the Sanquin Blood Supply Foundation.
Cold agglutinins are self-reactive antibodies that mistakenly bind to red blood cells when the body is exposed to cold temperatures. This binding causes red blood cells to clump together, or agglutinate, and to be marked for destruction, causing CAD symptoms.
Doctors use blood tests to measure two features of cold agglutinins: titer (levels in the blood) and thermal amplitude (the highest temperature at which the antibodies are active). Cold agglutinins with a high titer or a higher thermal amplitude are more likely to cause CAD symptoms. However, there is no universal agreement on how these tests should be performed, and different laboratories use different methods. This makes it difficult to compare results and guide treatment decisions.
Labs use different temperatures, practices
The team surveyed testing practices in 17 blood transfusion laboratories in the Netherlands.
They found that laboratories used a variety of testing strategies. Thirteen labs tested blood at cold temperatures — 4 C (39.2 F), 16 C (60.8 F), or both — while three tested across a wider range, including body temperature. One tested blood at room temperature. Labs also differed in sample type, using either plasma (the liquid part of the blood with clotting factors) or serum (plasma without clotting factors). Nearly all diluted blood samples with a saline solution before assessing cold agglutinin titer.
Some added bovine serum albumin (BSA), a protein that can improve the accuracy of the test by preventing antibodies from sticking to test tubes instead of red blood cells.
About half of the laboratories assessed cold agglutinin thermal amplitude at temperatures between 4 C and 37 C.
The researchers also tested blood samples from 16 people for cold agglutinins to see how differences in handling blood before and during testing may affect the results.
Fourteen of these people had a CAD diagnosis; the remaining two lacked information on their diagnosis. Cold agglutinin testing for all 16 participants was conducted at Sanquin.
When BSA was added to plasma samples, the tests often showed higher titers, suggesting that this method may be more sensitive. Subsequent analyses used BSA when diluting samples.
For 11 patients, titers were measured at 16 C using either plasma or serum. In six, results were similar between the two types of samples. The others showed slightly higher titers in serum or plasma. One sample had a much higher titer in plasma, but overall the differences were small.
Another finding was that blood samples collected normally and then reheated to body temperature (37 C) before testing (reheating samples) gave results similar to samples that were kept warm from the moment they were drawn (warmed samples).
Reheated plasma was also compared with standard plasma, or that kept at room temperature from the moment of collection. Reheating to body temperature generally gave equal or higher titers. Testing at different temperatures (4 C, 16 C, and higher) showed titers dropped as temperature increased, which is typical for cold agglutinins.
The team also analyzed samples from 100 healthy blood donors, 53 men and 47 women. Thirty had low titers of cold agglutinins that only reacted at 4 C, and six had cold agglutinins at 16 C. No cold agglutinins were found at 30 C (86 F).
In healthy people, these antibodies are usually linked to recent infections. They do not indicate disease unless they react at warmer temperatures or are abundant.
Overall, the results showed that testing for cold agglutinins is inconsistent across laboratories, and small changes in how blood is handled can affect results. Still, the researchers found practical ways to make testing easier and more reliable, such as using reheated samples and BSA.
While the number of samples in this study was relatively small, “our data provide practical insights,” the researchers wrote. “Standardizing the [blood] workup is a crucial step toward establishing evidence-based cutoffs for clinically relevant [cold agglutinin titers] and thermal amplitude in larger [groups] of patients.”
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