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with CD20 and DBA.44 immunostains, which are routinely used, and therefore IHC is readily avail- able. Finally, IHC can be used to quantitate the extent of MRD (Figure 1). Nevertheless, a number of limitations of IHC are apparent, including the fact that no single stain is specific, although combinations of tartrate resistant acid phosphatase (TRAP)/annex- in 1/cyclin D1 are highly specific and sensitive. Annexin 1 appears best, but also stains granulocyte precursors, sometimes making interpretation diffi- cult. The definition of MRD detected by IHC employed by the Northwestern University investiga- tors includes: (1) absence of HCL by routine
morphology; (2) presence of CD20þ or DBA.44þ cells in a number equal to or greater than the T-cell
population (CD45RO); (3) 450% of CD20þ or DBA.44þ cells exhibiting morphology of hairy cells. Immunophenotyping by FC has the important
potential advantages of being specific for a character- istic phenotype, detecting low levels of disease, and identifying hairy cells in the setting of leukopenia. However, it is important to note that the abnormal population of cells on occasion can be detected in the
bone marrow when the peripheral blood may be negative. Approximately 30% of patients with HCL have atypical immunophenotypes [9]. In a small study, Bengio and colleagues compared IHC (using anti-CD20 and DBA.44 detecting 1–10% positive cells with consistent morphology) and FC (any expression of CD11c/CD25/CD103 in the marrow or 40.3% of B-cells in the peripheral blood) to identify MRD [10]. Immunohistochemistry detected MRD in 46% compared to 64% for FC, but the number of patients was very small. Immunophenotyping has been reported to be
more sensitive than consensus-primer PCR [11]. However, with a new quantitative PCR assay (RQ- PCR) using patient-specific primer/probe combina- tions to surface immunoglobulin G (IgG) rearrange- ments, Arons and colleagues at the National Institutes of Health have shown that the latter is more sensitive (and clearly more specific) than immunophenotyping by FC, with a sensitivity of 1 in 1076 [7]. Few studies such as the latter report have directly compared methods. Patient-specific PCR is highly sensitive and specific. However, it is
Figure 1. Trephine core biopsy stained with H&E shows an interstitial infiltrate of small monocytoid lymphocytes with cytoplasm (A). These lymphocytes are positive for CD20, as shown in (B). These lymphocytes are not identifiable in the H&E stained section (C). However, the CD20 stain highlights small interstitial clusters of hairy cells remaining after therapy (D). Magnification (A–C) 6200, (D) 6400.
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