NK Cell Reference Ranges Established by Flow Cytometry in Reactive and Lymphomatous Lymph Nodes
Joseph Schumann, Alexandra M Harrington, Steven H Kroft, Horatiu Olteanu. MCW, Milwaukee, WI
Background: Reference ranges for NK cells in lymph nodes (LNs) were established 15 years ago from a limited number (<20) of cadaver donor specimens, and employed rudimentary flow cytometers and antibody combinations. Since it is important to have clinical reference ranges for NK cells in LNs, to help distinguish abnormal expansions of NK cells from reactive NK cell populations, we quantified by flow cytometry (FC) NK cells and other lymphocyte subsets in a large series of clinical specimens.
Design: 286 consecutive LN biopsies [138 reactive (RE); 148 lymphomas (LY)] were analyzed by FC on a FACSCanto II instrument, using antibodies against CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, CD38, CD45 and surface light chains. Lymphocyte subsets were identified by cluster analysis and values were calculated by standard statistical methods.
Results: There were 81 F/57 M (median age=48 years) in the RE LN group; 70 F/78 M (median age=63 years) in the LY LN group. Excised LNs showed: follicular and/or paracortical hyperplasia (110), necrotizing or granulomatous inflammation (22), angiomyomatous hamartoma and IgG4-related disease (3 each), follicular lymphoma (FL, 55), diffuse large B-cell lymphoma (DLBCL, 45), mantle cell lymphoma (11), marginal zone lymphoma (MZL, 11), chronic lymphocytic leukemia (CLL, 10), Hodgkin lymphoma (10), and peripheral T-cell lymphoma (6). Median and 5-95% ranges for T, B, and NK cell subsets are shown in Table 1. Median NK cells were significantly lower in LY than in RE LNs (p=0.0003). In the LY LN group, CLLs had a lower median percentage of NK cells (0.20%) than DLBCLs (0.64%, p=0.007), MZLs (0.51%, p=0.029) and low-grade (1 and 2) FLs (0.51%, p=0.002). B cells were more frequent in the LY cohort, reflecting an expanded neoplastic B-cell clone in the majority of cases.
Conclusions: We established updated clinical reference ranges for NK cells and other subsets in benign and lymphomatous LNs in a large cohort. This data can be used for assessment of NK cell expansions in LN specimens. We also found differences in the proportion of NK cells in RE vs. LY LNs, and between various LY categories, suggestive of a modulation of the NK cell-mediated immune response in those conditions.
|Reactive (n=138)||Lymphoma (n=148)|
|Subset (% of total lymphocytes)||Median||5-95%||Median||5-95%|
|Total B cells||29.82||9.25-66.73||59.05||14.46-96.85|
|Primary follicle/mantle zone B cells||26.57||7.24-51.95||N/A||N/A|
|Germinal center B cells||6.84||0.89-24.34||N/A||N/A|
|Total T cells||69.3||32.87-90.58||40.51||3.06-82.63|
|CD4(+) T cells||49.4||11.85-74.90||25.16||2.05-52.41|
|CD8(+) T cells||12.57||5.52-38.10||12.91||1.65-42.80|