Issue Highlights—May 2013

Taking the sting out of functional basophil testing

In this issue, we see manuscripts covering a wide assortment of topics of interest to clinical cytometrists. Among these excellent articles is one by Nullens and colleague (1) describing the measurement of intracellular histamine in basophils during the course of venom immunotherapy therapy (VIT). For individuals who suffer systemic reactions to wasp stings, VIT offers a specific treatment to reduce the odds of recurrent severe reactions (2). In this paper, a method to detect histamine release as well as cell surface markers of basophils is used to monitor the effects of VIT therapy. Although this study is limited in the number of patients studied, it offers examination of both function and phenotyping in rare cells as a method of monitoring the efficacy of a specific immunotherapy. Historically basophils were an often overlooked subset of leukocytes in the peripheral circulation, but in the past ten years, our ability to detect basophils and study their function has markedly increased (3–5). The readers are referred to several recent articles published in this journal for the latest developments in basophil assays in the clinical cytometry arena.

Doing the FOXP3 trot

Regulatory T (Tregs) cells are key components in down-regulating immune responses, and have been shown to play a role in diseases ranging from cancer to autoimmune diseases and infections (6). Tregs are CD4 T cells most often characterized by the expression of forkhead box P3 (FoxP3). These cells also regarded as having have bright expression of CD25 and low expression of CD127, although a recent publication has shown a great heterogeneity in the phenotypes of Tregs found in patients with chronic lymphocytic leukemia (7). In this issue, Demaret et al demonstrate a one step technique for staining FoxP3 in order to identify these cells (8). FoxP3 is an intracellular antigen, and as such, fixation/permeabilization techniques are required for staining. In the approach presented here, a commercial no wash kit is used for staining these cells with FoxP3 as well as surface markers with good results. This expedited protocol might obviate the need to use surrogate staining, such as CD25high and CD127 low on CD4 cells to identify Tregs (9). One might hope that this expedient method would facilitate samples in clinical trials being run on fresh rather than cryopreserved samples, as cryopreservation has been shown to negatively impact the detection and quantification of Tregs (10).

Seeing double trouble

One of the most common uses of flow cytometry in the clinical laboratory is for the detection of leukemic cells in the bone marrow or peripheral circulation. While clinicians most often suspect a single lineage of leukemic cells, it is not altogether uncommon for the cytometry results to reveal a bilineal, or mixed phenotypic acute leukemia (MPAL). In the current issue, Rahman and colleagues report an unusual case of a patient presenting with both T and B lineages among the leukemic blasts (11). Even among MPAL this phenotype is rare, with most cases displaying a myeloid component in addition to the lymphocytic lineage (12, 13). Those who find the current case report of interest might also want to review a previous case report in which three simultaneous lymphoproliferative disorders (HCL, B-CLL, and T-LGL) were detected in both peripheral blood and bone marrow (14).

More double trouble

Double hit lymphomas (DHL), those with concurrent translocations of MYC and BCL2are rare, but well documented clinical entities (15). The issue of whether or not flow cytometry can be of use in identifying these lymphomas is the subject of investigation by Platt et al (15). Phenotypic abnormalities have been associated with DHL, including dim expression of CD20 and/or CD19. In the current report, the authors find these abnormalities, but not in such frequencies to permit these to be used as unequivocal biomarkers of DHL. Rather, they suggest that the immunophenotypic findings be used only in conjunction with clinical and other laboratory findings to recommend cytogenetic testing. A case of double hit lymphoma was presented as a ‘Case Study Interpretation (CSI)’ during the annual meeting of the International Clinical Cytometry Society in 2011 for attendees to test their diagnostic skills. For those who were unable to attend, this case is available in this journal (16).

J. Philip McCoy Jr., PhD

Bethesda, MD