Assessment of bone marrow plasma cell infiltrates in multiple myeloma: the added value of CD138 immunohistochemistry.

Assessment of bone marrow involvement by malignant plasma cells is an important element in the diagnosis and follow-up of patients with multiple myeloma and other plasma cell dyscrasias. Microscope-based differential counts of bone marrow aspirates are used as the primary method to evaluate bone marrow plasma cell percentages. However, multiple myeloma is often a focal process, a fact that impacts the accuracy and reliability of the results of bone marrow plasma cell percentages obtained by differential counts of bone marrow aspirate smears. Moreover, the interobserver and intraobserver reproducibility of counting bone marrow plasma cells microscopically has not been adequately tested. CD138 allows excellent assessment of plasma cell numbers and distribution in bone marrow biopsies. We compared estimates of plasma cell percentages in bone marrow aspirates and in hematoxylin-eosin- and CD138-stained bone marrow biopsy sections (CD138 sections) in 79 bone marrows from patients with multiple myeloma. There was a notable discrepancy in bone marrow plasma cell percentages using the different methods of observation. In particular, there was a relatively poor concordance of plasma cell percentage estimation between aspirate smears and CD138 sections. Estimates of plasma cell percentage using CD138 sections demonstrated the highest interobserver concordance. This observation was supported by computer-assisted image analysis. In addition, CD138 expression highlighted patterns of plasma cell infiltration indicative of neoplasia even in the absence of plasmacytosis. We conclude that examination of CD138 sections should be considered for routine use in the estimation of plasma cell load in the bone marrow.

DRAQ5-based DNA content analysis of hematolymphoid cell subpopulations discriminated by surface antigens and light scatter properties.

BACKGROUND: Analysis of cell cycle kinetics offers important information regarding the behavior of normal and neoplastic cells. Most often, cell cycle determinations by flow cytometry (FCM) have been performed using whole-sample analysis with intercalating dyes like propidium iodide (PI). The cell cycle phase assessment in individual cell subsets in heterogeneous samples is best performed using combined antigen/scatter and DNA analysis. DRAQ5, a novel DNA binding dye that excites at 488 nm and emits in the far red spectra, rapidly penetrates intact live cells while preserving their light scatter properties and expression of surface antigens. We evaluated the ability of this dye to measure cell cycle phases in a variety of clinical hematolymphoid samples. METHODS: We first compared whole sample DRAQ5 and PI cell cycle analyses in 26 clinical hematolymphoid samples. Next, we analyzed cell subpopulations in 39 samples of nonpathologic bone marrow by performing simultaneous CD45/CD34 and DRAQ5 staining. We assessed cell cycle characteristics specific to each population identified by CD45/CD34/side light scatter: lymphocytes, monocytes, immature and mature granulocytes, nucleated erythroid cells, and early precursors. RESULTS: Whole sample DNA cell cycle analyses by DRAQ5 and PI showed no significant differences in S-phase. DRAQ5, however, produced slightly larger coefficients of variation. DRAQ5-based DNA content analysis was easily performed on the distinct marrow cell subpopulations, since light scatter and antigen expression were completely preserved. Significant differences in S-phase were noted between subpopulations of cells exhibiting different degrees of maturation. CONCLUSIONS: Because of its simplicity of use, excitability with 488 nm lasers, and the ability to stain viable cells, DRAQ5 should prove most useful in the kinetic evaluation of normal and neoplastic hematolymphoid cell subsets identified by light scatter and antigenic expression.

Detection of T-regulatory cells has a potential role in the diagnosis of classical Hodgkin lymphoma.

Previous studies have demonstrated an increase in T-regulatory cells in the involved lymph nodes and peripheral blood of patients with Hodgkin lymphoma. Our study examined whether the detection of T-regulatory cells by flow cytometry could distinguish classical Hodgkin lymphoma (CHL) from benign cases and B-cell non-Hodgkin lymphomas (B-NHL). We measured CD4, CD25, and CD152 in 14 CHLs, 2 nodular lymphocyte-predominant Hodgkin lymphomas, 31 B-NHLs, and 54 benign cases. All T-regulatory cell parameters, including percent lymphocytes CD4+/CD152+ and CD4+/CD25+/CD152+, and mean and median CD152 expression in CD4+/CD25+ lymphocytes, were higher in CHL than in B-NHL and benign. Mean CD152 in CD4+/CD25+ lymphocytes distinguished CHL from benign with 79% sensitivity and 100% specificity, and from B-NHL with 71% sensitivity and 90% specificity. Overall, our results show that T-regulatory cells are increased in CHL and their detection may be a useful tool in differentiating CHL from other entities.