[1402] CD335 (NKp46) Specifically Identifies Natural Killer Cells by Diagnostic Flow Cytometry

Aharon G Freud, Sibing Wei, Gary M Gitana, Yasodha Natkunam, Susan K Atwater. Stanford University, Stanford, CA

Background: CD335 (NKp46) is a surface activation receptor that is expressed on natural killer (NK) cells. Few studies have reported aberrant CD335 expression on T cell neoplasms; however, the clinical application of CD335 in routine diagnostic flow cytometry is unknown.
Design: A 6-color cocktail containing CD3 APC-H7, CD16 V450, CD45 PerCP, CD56 FITC, CD161 (NKRP1A) APC, and CD335 PE antibodies was optimized and evaluated on patient specimens submitted for diagnostic flow cytometry. Patient samples were processed and analyzed by clinical laboratory scientists. Populations were gated using CD45 expression and forward and side scatter characteristics. Diagnoses were made using WHO 2008 criteria.
Results: A total of 223 specimens, including blood (n=71), bone marrow (n=103), and lymph node (n=32), were stained with the 6-color antibody cocktail. Diagnoses included reactive conditions as well as B cell, T cell, plasma cell, and myeloid neoplasms.

CD335 expression by flow cytometry

	Percent of cases with positive CD335 expression
Diagnosis	Neoplastic/atypical population	Background T cells	Background NK cells
Reactive (n=124)	NA	1.6%	100%
Atypical T or B lymphocytosis (n=17)	0%	0%	100%
Atypical NK lymphocytosis (n=3)	66.7%	0%	100%
B lymphoblastic leukemia (n=4)	0%	0%	100%
B cell leukemia/lymphoma (n=35)	0%	0%	100%
T lymphoblastic leukemia (n=3)	0%	0%	100%
T cell leukemia/lymphoma (n=16)	12.5%	0%	93.8%
Plasma cell dyscrasia (n=7)	0%	0%	100%
Myeloid neoplasm (n=14)	0%	0%	100%

In 218 of 222 cases with detectable CD335 expression, the latter was restricted to NK cells. In contrast, other NK-associated antigens were often expressed by subsets of T cells (CD56 and CD161) and monocytes (CD16 and CD56). Three atypical NK lymphocytoses lacked CD335 on the majority of the atypical NK cells. Two atypical T cell proliferations showed minor subsets (<10% of T cells) expressing CD335. One T-cell large granular lymphocytic leukemia expressed CD161 and dim CD335 but not CD16 or CD56. One ALK+ anaplastic large cell lymphoma expressed dim partial CD56 and dim CD335 but not CD161. Overall, the sensitivity and specificity of CD335 labeling of NK lineage cells was 99.6% and 98.2%, respectively.
Conclusions: The 6-color flow cytometry cocktail that we utilized detects NK cell derived proliferations with very high sensitivity and specificity. We show that CD335 is thus far the most lineage-specific marker for NK cells, and assessment of its expression may be useful to identify and enumerate NK cells in clinical specimens. The observation that CD335 may be aberrantly expressed on neoplastic T cells further supports its potential utility in diagnostic flow cytometry.
Category: Hematopathology

Monday, March 19, 2012 9:30 AM

Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 230, Monday Morning