A 57-year-old man presented with proteinuria and was diagnosed with nephrotic syndrome. Physical examination revealed no signs of purpura or other dermatological manifestations. An alert, abnormal distribution of leukocyte distribution was triggered by an automated hematology analyzer (DxH 900, Beckman Coulter) in the clinical laboratory. The white blood cell (WBC) count was 4.1 × 109/L, with an absolute neutrophil count of 1.5 × 109/L. The analyzer flagged a possible left shift and the presence of immature granulocytes. Red blood cell count was 3.5 × 1012/L, and platelet count was 296 × 109/L. As shown in Figure 1a, the WBC scattergram demonstrates poor cell clustering, and the corresponding histogram exhibits a bimodal distribution. An extensive agglutination of leukocytes was observed in the peripheral blood smear prepared from EDTA-anticoagulated blood. The agglutination was predominantly composed of mature neutrophils, mainly localized at the edges of the smear, with occasional clusters of lymphocytes also present (Figure 1b). In addition, erythrocytes were observed to aggregate in a rouleaux formation, and no platelet clumping was detected. Agglutination was still observed when blood smears were prepared using citrate-anticoagulated blood (Figure 1c) and fresh capillary blood collected from the finger prick without anticoagulant (Figure 1d). Therefore, an anticoagulant-induced mechanism was excluded. To investigate the possibility of cold-induced agglutination, a rewarming experiment was conducted. Upon incubation of EDTA-anticoagulated blood at 37°C for 30 min, the WBC count was 5.7 × 109/L, with a neutrophil count of 3.8 × 109/L. The degree of aggregation markedly diminished, with only a few residual neutrophil clusters remaining (Figure 1e), suggesting a temperature-dependent process. A more interesting phenomenon has caught our attention. The EDTA-anticoagulated blood exhibited a gelatinous appearance when stored at 4°C for 15 min (Figure 1f), then reverted to a liquid state upon warming without any visible precipitate or granularity (Figure 1g). This behavior is consistent with the characteristics of cryoglobulinemia-induced coagulation. To investigate the underlying etiology, an extensive diagnostic evaluation was performed. Serum immunoglobulin quantification revealed a markedly elevated IgM level of 13 g/L. Serum and urine immunofixation electrophoresis demonstrated the presence of a monoclonal protein of the IgM kappa type. Qualitative testing for cryoglobulins was positive, monoclonal IgM kappa type. Bone marrow examination showed 3.5% plasma cells on smear. CD19-positive B cells accounted for 60.3% of lymphocytes, with suspected kappa light chain restriction by flow cytometric analysis. Vacuolar degeneration of the glomerular basement membrane, and focal subepithelial fuchsinophilic deposits were identified in renal biopsy tissue. Scattered infiltrates of small, morphologically uniform lymphoid cells were observed in the renal interstitium. Congo red staining was negative for amyloid deposition. Immunohistochemical analysis demonstrated strong C4d positivity with granular deposition along the glomerular capillary walls, while staining for phospholipase A2 receptor was negative. The scattered and nodular aggregates predominantly composed of small B lymphocytes in the interstitial. Immunohistochemistry showed positivity for CD20 (+++) and Bcl-2 (+++), and kappa light chains were expressed in a scattered pattern. The final diagnosis of monoclonal gammopathy of renal significance (MGRS) was established based on the presence of monoclonal IgM kappa and the pathological evidence from both bone marrow and renal biopsy specimens. The monoclonal IgM kappa exhibited cryoglobulin and cold agglutinin-like properties, resulting in the formation of gelatinous material and neutrophil agglutination in the complete blood count sample. The patient initially received a 100 mg infusion of rituximab, followed by a second dose of 500 mg 6 months later. Three months after the completion of the second infusion, follow-up evaluation showed cryoglobulin testing was negative, urinary protein levels were reduced, and EDTA-anticoagulated blood smear showed no evidence of leukocyte agglutination (Figure 1h). Peripheral blood leukocyte agglutination is a rare laboratory phenomenon that poses significant challenges to accurate white blood cell quantification and may cause pseudoneutropenia 1. The DxH 900 hematology analyzer utilizes hydrodynamic focusing technology to measure cellular volume, electrical conductivity, and multi-angle light scatter signals (VCS) simultaneously, enabling WBC counting and differential classification. When neutrophil agglutination occurs, aggregated cells may be detected as single analytical events, resulting in aberrant cellular parameter measurements. This phenomenon leads, on the one hand, to poorly defined clusters on the WBC scattergram and subsequent misclassification. On the other hand, neutrophil aggregates exhibit an apparently increased cellular volume that may exceed the analyzer's predefined WBC volume analysis window, causing the affected cell population to disappear from the WBC volume histogram and ultimately triggering instrument flags or alarms. The proposed mechanism includes the presence of EDTA-dependent antibodies and temperature-sensitive antibodies, especially IgM antibodies. The monoclonal IgM kappa is implicated in > 90% of cold agglutinin disease cases—typically inducing erythrocyte agglutination at 4°C and autoimmune hemolytic anemia 2. A previously published report documented a case of cold agglutinin disease associated with neutrophil aggregation 3. However, this case represents the first documented co-occurrence of type I cryoglobulinemia, MGRS, and cold-induced leukocyte agglutination. This triad indicates that monoclonal IgM may directly promote leukocyte aggregation via cryoprecipitation or antibody-mediated crosslinking. The observed resolution of symptoms following rituximab-based therapy aligns with established management principles for clinically significant monoclonal gammopathies 4. Rituximab selectively targets CD20-positive B-cell clones, thereby suppressing pathogenic IgM production—a mechanism that further supports the antibody-mediated etiology in this case. All authors contributed to the paper's conception and design. Clinical and histological data were collected by Yu Yang, Yating Li, and Ziyi Zeng. The draft of the manuscript was written by Yu Yang, Chenxue Qu, and Meiling Wang, and all authors read and approved the final manuscript. This study was approved by the Medical Ethics Committee of Peking University First Hospital. The written informed consent for publication was obtained from the patient. The authors declare no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request.
Yu et al. (Sat,) studied this question.