Heavy/Light Chain Assay Useful Marker in Cold Agglutinin Disease, Study Suggests

Current standard modalities for detecting and quantifying monoclonal immunoglobin in patients with cold agglutinin disease lack adequate sensitivity. Heavy chain/light chain assay may be more effective.

There is room for improvement among the assays typically used to characterize cold agglutinin disease (CAD), especially where sensitivity is concerned.

A letter to the editor published in British Journal of Haemotology suggests an alternative assay, heavy chain/light chain (HLC), as a more effective technique to detect and quantify monoclonal immunoglobin (Ig) in patients with CAD.

Primary CAD is rare, affecting 15% of patients with autoimmune hemolytic anemia (AIHA), and cases typically feature the monoclonal cold agglutinin immunoglobin M (IgM). This suggests clonal proliferation of cells from a lymphoproliferative disorder, such as monoclonal gammopathy of undetermined significance (MGUS), lymphoplasmacytic disorder, or marginal zone lymphoma. The assays currently used for monitoring the monoclonal component of CAD—serum protein electrophoresis (SPE) and immunofixation (IFX) assays—each have shortcomings, presenting a need for a sensitive and quantitative strategy for monitoring monoclonal Ig. This is particularly important because immunochemical response can precede clinical response.

HLC (Hevylite; Binding Site) identifies and quantifies monoclonal Ig, including IgM, IgG, or IgA. It is based on Ig binding to junctions between a kappa or lambda light chain and its heavy chain partner, separately quantifying IgMk and IgMλ and calculating a ratio to determine whether monoclonality is abnormal. The assay has been found useful in IgA myeloma, and the current study aims to gauge its efficacy compared with SPE and IFX for monitoring monoclonal IgM in patients with CAD.

The retrospective study included all patients with CAD at the Saint-Louis Hospital in Paris from 1993 to 2018. Inclusion criteria included hemolysis, monospecific direct antiglobulin test (DAT) results strongly positive for complement component C3d, and a positive cold agglutinin titer at 4°C.

Forty-eight patients were included in the study, with a median age of 62 years. Underlying MGUS, lymphoplasmacytic disorder, and marginal zone lymphoma were identified in 63.41%, 17.70%, and 12.20% of patients, respectively. Nearly half (44.74%) of patients experienced acrocyanosis, and 15.38% had adenopathy and/or splenomegaly. Overall, 87.5% tested positive for monospecific anti-C3d antibody on DAT, while the rest were strongly positive for anti-C3d and weakly for IgG antibody. Treatment was used for 66.6% of patients, with most undergoing rituximab-based therapy regimens.

All patients in the study underwent SPE, IFX, and HLC assays. The SPE assay was positive in 18 patients, IFX showed monoclonal IgM in 25 patients (2 IgMλ and 23 IgMk), and the HLC testing showed abnormal ratios in 32 patients (25 IgMk and 7 IgMλ). Both the IFX and SPE assays showed a monoclonal IgM component in 15 patients, and HLC identified IgM that was missed by IFX in 8 patients. HLC also detected IgM that SPE missed in 17 patients.

Overall, the HLC ratio was more sensitive than SPE or IFX at detecting the IgM monoclonal component in patients with CAD. When patients were treated with rituximab, HLC ratios decreased with treatment and indicated the subsequent decrease of the monoclonal component.

“SPE and IFX are good biomarkers to help clinicians but lack sensitivity, especially in CAD, since IgM is often low and difficult to detect. In our study, HLC detects more frequently the monoclonal component than usual assays (SPE and IFX),” the authors wrote. “For 5 patients who were followed with this assay, HLC was useful to track the monoclonal component after treatment and was consistent with clinical improvement.”

Despite study limitations, including its retrospective nature and small size, as well as a lack of consistency regarding the timing of HLC testing, the study authors conclude that HLC is a useful marker for detecting and monitoring the monoclonal component of primary or secondary CAD.


Ursule-Dufait C, Bengoufa D, Theodorou I, Villesuzanne C, Arnulf B. Heavy chain/light chain assay is a useful biomarker for diagnosis and management of patients with cold agglutinin disease. Br J Haematol. Published online June 22, 2022. doi:10.1111/bjh.18317