Representing 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 Fc 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 Fc 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 + 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 (CD19+/CD10+), small lymphocytic leukemia/lymphoma and mantle cell lymphoma (CD19+/CD5+), etc). This allows not only for immunophenotypic demonstration of light chain restriction, but also assists in further subclassifying the type of NHL/chronic leukemia involving the bone marrow. Differential diagnosis of bone marrow hematogones from surface immunoglobulin negative malignant B cells was not mentioned in the article by Naughton et al. The article states that “flow cytometric results were considered positive if ≥50% gated B-cells lacked surface immunoglobin, CD19, or CD20.”1 Bone marrow hematogones are immature lymphoid cells of B-cell origin that characteristically lack surface immunoglobulins but are CD19+ and may coexpress CD20. Hematogones are not malignant, but their number may increase markedly in reactive, postinfectious, postchemotherapy restaged bone marrow. It is virtually impossible to differentiate these hematogones from neoplastic and surface immunoglobulin negative cells of follicular center cell lymphoma without four color CD19/CD10/κ/λ analysis. Representing a Canadian healthcare provider, we also cannot help but comment on the cost of the flow cytometric analysis in the published article. The authors quote the amount of 1720 1995 $US per case for a panel of 14 antibodies. For a 25% of this quoted amount our laboratory performs more sophisticated chronic leukemia/NHL workup panels that are comprised of twice as many antibodies. Our costs, which amount to 450 $Cdn, include reagent (33 antibody panel), and technical and professional fees. Traditional staging of a bone marrow aspirate and biopsy in our institution is estimated to cost 300 $Cdn (including nursing, technical, and professional fees). The above cost analysis makes flow cytometry a very sensitive and yet cost-effective method of staging NHL in our center. In contrast to the published article, our laboratory has yet to see a single case that was considered positive by a standard morphologic assessment of bone marrow aspirate and biopsy and yet negative on flow cytometry. However, we do encounter cases routinely that are designated as either morphologically negative, atypical, or showing the presence of “nonspecific lymphoid aggregates” that, without immunophenotypic analysis, would spuriously have been considered negative, missing the actual minimal residual disease. If appropriately performed and interpreted, flow cytometric analysis may indeed replace the traditional method of staging NHL patients. Iwona Auer M.D.*, Joanne Luider B.Sc.*, * Flow Cytometry Laboratory Calgary Laboratory Services Foothills Medical Centre, Calgary, Alberta, Canada
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Open Access
Jan 1, 2001
Hideyuki Kawabata + 4
