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. 2025 Sep;120(9):944-949.
doi: 10.1111/vox.70069. Epub 2025 Aug 4.

Unifying serological testing for cold agglutinins

Marit Jalink  1   2 , Femke V M Mulder  1   3 , Marianne E George  4 , Josephine M I Vos  4   5   6 , Masja de Haas  1   4   7 , Claudia C Folman  4 ; DRAIHA Consortium
Collaborators, Affiliations

Unifying serological testing for cold agglutinins

Marit Jalink et al. Vox Sang. 2025 Sep.

Abstract

Background and objectives: Cold agglutinins (CAs) are immunoglobulin M autoantibodies that optimally bind to red blood cells at low temperatures. The clinical significance of CAs is usually characterized by the CA titre and thermal amplitude. However, there is no consensus on the optimal testing strategy. In this study, we sought to unify CA testing.

Materials and methods: A survey on CA evaluation was conducted in 17 Dutch transfusion laboratories. Additionally, samples from 16 patients with CAs were used to evaluate (pre)analytical variations in test outcomes.

Results: The survey on CA evaluation revealed heterogeneity in the indications for CA workup and in (pre)analytical conditions. Titration results between EDTA plasma and serum did not differ. However, titres were higher when 2% bovine serum albumin (BSA) was present in the titration medium. Routinely collected EDTA-blood kept at 37°C for 10 min before plasma isolation (reheated) showed equivalent results to plasma from blood kept at 37°C from collection through separation.

Conclusion: This study showed that different (pre)analytical conditions influence test outcomes in the characterization of CAs. Importantly, CA testing can effectively be performed with reheated blood samples, which will facilitate a more practical workflow for laboratories. International standardization of CA testing for diagnostic use is essential.

Keywords: autoimmune haemolytic anaemia; cold agglutinin disease; cold agglutinins.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Comparison of test variables in cold agglutinin (CA) titration at 16°C. (a) EDTA‐anticoagulated plasma titrated in either phosphate‐buffered saline (PBS) or PBS with 2% bovine serum albumin (BSA). (b) Serum and EDTA plasma (in PBS with 2% BSA). (c) Standard EDTA plasma versus reheated EDTA plasma (in PBS with 2% BSA). Titres were expressed as 2 X , where X represents the number of twofold serial dilutions (1:64 = 26, 1:16,000 = 214).
FIGURE 2
FIGURE 2
Cold agglutinin (CA) titration in warm blood samples were compared with CA titres in reheated EDTA‐anticoagulated plasma of the same patients.
See this image and copyright information in PMC

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