Is Flow Cytometry a Useful Test? R epresenting a busy flow cytometry laboratory with an annual workload of approximately 1400 acute leukemia and malignant lymphoma cases, we read the article by Naughton et al. 1 with considerable interest. The authors concluded that "flow cytometry of bone marrow aspirates has a limited role in the routine staging and follow-up of patients with an established diagnosis of [non-Hodgkin's lymphoma] NHL." 1 However, this conclusion must be challenged because a number of methodologic flaws and inadequacies in the process, analysis, and interpretation are evident in this study. 2,3 The authors used Ficoll-Paque (Pharmacia Biotech, Piscataway, NJ) processed specimens rather than recommended erythrocyte lysis. 2 Ficoll-Paque centrifugation of bone marrow aspirates is known to result in the selective loss of some cell populations. This phenomenon is related to the fact that neoplastic lymphoid cells may have a different buoyant density from normal lymphocytes and may not be found where anticipated in the gradient. 2 The authors, possibly unknowingly, thus may have reduced the number of potentially analyzable malignant lymphoid cells in their samples, which may explain a portion of their staged cases with a negative flow result.
Single color flow cytometry was used on 81 specimens and two color flow cytometry on the remaining 192 specimens. It currently is widely accepted that even two color flow cytometric analysis is inadequate for an appropriate characterization of hematologic neoplasia and virtually precludes an accurate assessment of minimal residual disease. Five parameter analysis including forward and side scatters accompanied by at least three color analysis should constitute the minimal standard for immunophenotypic analysis of lymphoproliferative diseases, with a sensitivity approaching detection of one malignant cell in 100,000 reactive lymphocytes. 2,3 The authors make no mention of attempted blockade of cellular F c receptors. This step is to eliminate the common error of spuriously detecting light chain restriction while in fact observing a phenomenon of passive cytophilic immunoglobulin absorbed onto nonlymphoid B cells. In contrast, failed blockade of F c receptors on lymphoid cells may lead to false-negative results.
Apparently no specific gating strategies were utilized. For increased sensitivity, the routine use of a B-cell gate (CD19 ϩ lineage gate) is recommended. In addition, application of other unique markers known to be present on subsets of non-Hodgkin's lymphoma (NHL) and chronic leukemia cells provides added resolution for the assessment of bone marrow involvement. Unique gating strategies should differ based on the type of NHL present at diagnosis (i.e., follicular lymphoma (CD19ϩ/CD10ϩ), small lymphocytic leukemia/ lymphoma and mantle cell lymphoma (CD19ϩ/CD5ϩ), etc).