A b s t r a c t The diagnosis of classical Hodgkin lymphoma (CHL) FC, often present as T-cell-HRS-cell rosettes. In this study, we examined the usefulness of a novel 9single tube FC assay to diagnose CHL in lymph nodes. We used the FC assay to determine diagnostic sensitivity and specificity in 279 blindly identified and 141 selected (for specimen type or cytopreparation morphologic features suggesting CHL) tissues. Of the 53 morphologically defined CHL cases identified (10 in the unselected

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... up; 43 in the selected group), the FC assay diagnostic sensitivity and specificity were 88.7% and 100%, respectively. With the current availability of 8 (or more) color clinical flow cytometers, this assay can now be applied to routinely immunophenotype and confirm a diagnosis of CHL or as an adjunct to immunohistochemical analysis. has been made in tissue sections as attempts to identify neoplastic Hodgkin and Reed Sternberg (HRS) cells in lymph nodes by flow cytometry (FC) have been unsuccessful. However, we have recently demonstrated that HRS cells can be identified by Classical Hodgkin lymphoma (CHL) is a unique type of B-cell lymphoma 1-3 in which the neoplastic Hodgkin and Reed-Sternberg (HRS) cells are infrequent 2,4 (usually <1% of the leukocytes in a lymph node), have an unusual immunophenotype (CD30+ and CD15+ and CD3-, CD20-, and CD45-, features often used to confirm the diagnosis in tissue sections 1,4,5 ), have characteristic morphologic features, 6,7 and demonstrate rosettes of T cells bound to an HRS cell. [8] [9] [10] [11] [12] [13] The HRS cells are embedded in an inflammatory, nonneoplastic infiltrate that includes lymphocytes, histiocytes, plasma cells, and eosinophils. 1, 4, 6 Although flow cytometry (FC) is useful in the diagnosis of many hematopoietic neoplasms and can routinely detect very small populations (often <0.01% of the leukocytes), 14 its usefulness in contributing to the diagnosis of CHL involving lymph nodes has been limited. Although most FC studies of CHL have looked at changes in the composition of reactive lymphocytes, such as the CD4/CD8 ratio of the associated T cells, 15 the HRS cells have generally escaped detection by this technique. We recently described a method of identifying HRS cells in lymph nodes by FC using a 2-tube, 10-color assay that used a combination of antibodies reactive with antigens on HRS cells (CD15, CD30, CD40, CD71, and CD95), B cells (CD19 and CD20), T cells (CD3 or CD5), macrophages (CD64), and leukocytes (CD45). 16 It is interesting that a subset of the HRS cells in most cases examined demonstrated expression of T-cell antigens (CD3 and CD5) and bright CD45 owing to the presence of HRS-cell-T-cell rosettes (aggregates). Preincubation with unlabeled monoclonal antibodies to the adhesion molecules CD2 and LFA-1 (present on T cells 12 ) and CD54 and Upon completion of this activity you will be able to: • describe the flow cytometry derived immunophenotype of Hodgkin and Reed Sternberg (HRS) cells in classical Hodgkin lymphoma (CHL). • discuss the phenomenon of T cell-HRS cell rosettes and its influence on the CHL immunophenotyping. • compare and contrast the immunophenotypes in CHL with those seen in anaplastic large cell lymphoma, T cell-rich large B-cell lymphoma, and nodular lymphocyte-predominant Hodgkin lymphoma. • describe how the HRS cells differ immunophenotypically from reactive CD30-positive immunoblasts. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this educational activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 442. Exam is located at www.ascp.org/ajcpcme.