Rapid Immunoprofiling of New Lymphocytosis Cases Using a Routine Hematology Analyzer: A Multicenter Study.
Authors: Adriaansen Henk , van den Berg Gita A , Herruer Martin H. , Schuitemaker Joost , Smit Jan W. , van Weel Tanja , Hoffmann Johannes J 
Published: April 2020

Abstract
Introduction: Lymphocytosis is a frequent finding in the routine hematology laboratory. Most often, lymphocytosis is caused by a reactive increase of T-, B- and/or NK-lymphocytes, but it may represent the first sign of a malignant monoclonal B-cell disorder as well. Currently, it is common practice in mild and moderate lymphocytosis to follow up the patient for three months in order to see whether this condition is chronic. If the lymphocytosis proves to be persistent, immunophenotyping is necessary to establish whether a reactive or a malignant disorder is causing it. It is not unusual that patients become concerned during this period as to the nature of their disease. Although the finding of lymphocytosis and/or instrument flagging may result in a manual differentiation and probably in detecting morphologic abnormalities in the lymphocytes, direct immunophenotyping is preferable to a manual differentiation, since morphology is not sufficiently sensitive and specific. However, immunophenotyping is time-consuming, expensive and until now only available in specialized laboratories. A rapid immunological screening (immunoprofile) could help reducing the number of extensive immunophenotypes. Moreover, patients with newly found lymphocytosis and their physicians may appreciate to have a more rapid indication on the nature of the lymphocytosis. Therefore, we studied the feasibility of a rapid immunoprofile, using a routine hematology analyzer, immediately after a new lymphocytosis case was detected. It was not our goal to provide a final immunophenotype, but rather an indication whether the lymphocytosis is reactive or potentially malignant. In the latter case, a full immunophenotype would still be necessary.
Methods: Five hospital laboratories investigated all samples with previously unknown lymphocyte count &gt; 5.0 109/L or a variant lymphocyte flag from patients over 12 years of age. The immunologic analysis was performed on the same blood sample within 24 hours from collection. Six pairs of monoclonal antibodies were used: CD3/4, CD3/8, CD3/19, CD3/16+56, CD3/5 and CD3/HLA-Dr (each pair FITC/PE labeled, respectively; IQ Products, Groningen, the Netherlands). The analysis was carried out using the fully automated CELL-DYN Sapphire hematology analyzer (Abbott Diagnostics, Santa Clara, CA, USA) and FCS files were collected. Data analysis of the FCS files was done centrally using commercial flow cytometry software (WinList; Verity Software House, Topsham, ME, USA).
Results: In total, the five laboratories analyzed 111 patient samples meeting the inclusion criteria. Of these patients, the majority showed a mixed pattern of increased T-cells together with B- and/or NK-cells (n=59; 53%). In 14 patients (13%), only T-cell counts were increased; no patient had increased NK-cells only and 34 patients (31%) showed increased B-cells. The remaining 4 patients (4%) could not be classified unambiguously. Of the 34 patients with increased B-cells, 25 had approximately equal numbers of CD5+ and 19+ lymphocytes, which is highly suggestive of a CLL phenotype. There were no cases in which the lymphocytosis would have been classified differently, if the HLA-Dr antibody had been omitted. Thus, the immunoprofile can be further limited without losing information as to the nature of the lymphocytosis.
Discussion: The majority of patients in our study (73; 66%) had reactive lymphocytosis. In addition, 25 patients (23%) had CD5+, CD19+ B-cell lymphocytosis, suggestive of CLL or monoclonal B-cell lymphocytosis. The latter group needs additional immunological investigation and further clinical follow-up. By using this immunoprofile screening strategy, the number of full immunophenotypes could be reduced by approximately two thirds, which means a significant reduction in workload and cost to the laboratory.
Conclusion: We have shown that it is well feasible to rapidly discriminate reactive from potentially malignant causes in patients with newly established lymphocytosis. The incidence of lymphocytosis with CLL-like phenotype amounted to 23% in our study, which is higher than reported so far in the literature.