Issue highlights—March 2013
DIFFERENT APPROACHES IN MRD
Multicolor flow cytometry was quite a technical novelty some years ago but today it became a daily diagnostic service in several flow cytometry laboratories. The publication of Sartor and Gottlieb in this issue of the journal (1) utilizes the advancement in flow technology and the wide array of reagent availability and they described a 10 color single tube flow cytometry assay to optimize the accuracy and sensitivity of minimal residual disease (MRD) detection. One particular problem in detecting MRD is the occurrence of cytopenia that may hamper the identification of residual cells when analyzing several tubes. Placing all of the informative markers in one tube reduces the total number of lymphocytes required for analysis in patients with lymphopenia following chemotherapy and facilitates direct exclusion of contaminating T cells during cell analysis. The assay was established and intended for use only by reference laboratories experienced in multi-parameter flow cytometry for CLL. This way of MRD detection, however also requires advanced instrumentation and thus cannot be achieved in several smaller laboratories. Literature from developing countries addressing therapeutic or laboratory practices related to MRD, is largely lacking, as in many resource-poor countries this advanced type of MRD detection is not feasible. Patkar and coworkers in their first paper from India published last year in this journal (2) described their experience in establishing a flow cytometry-based MRD assay for precursor B lineage ALL (BCP-ALL) with emphasis on assay standardization and cost. They utilized a 4-color approach that allowed MRD analysis at diagnosis and at two follow-up time points to be done in total for $100–150 and they found that this cost-effective MRD panel was applicable to over 90% of patients.
ZAP-70: A TEST FAR FROM BEING PERFECT
ZAP-70 testing made a long journey since its original description 10 years ago (3) but it seems to be an assay that is constantly being upgraded and improved since subtle changes in measurement conditions as well as data analysis may considerably influence the results. In the paper by Rizzo et al. in this issue of the journal (4) a potential methodological bias is highlighted by the authors on reporting ZAP-70 values in B-CLL. In the analysis of this intracellular protein it is usually agreed that phycoerythrin-conjugated (PE-conjugated) SBZAP monoclonal antibody (mAb) is one of the best reagents for this method. Currently most groups express flow cytometric results of ZAP-70 expression in CLL B-cells as a ratio between mean fluorescence intensities (MFI) of ZAP-70 in CLL B-cells and in normal residual or externally added B-cells or, more frequently, as a ratio between ZAP-70 MFI values in residual T-cells and CLL B-cells (ZAP-70 T/B ratio). The main disadvantage of the former method is that residual normal B-cells may be virtually absent in peripheral blood of patients with CLL and external addition of normal B-cells to the blood sample prior to staining supposes that ZAP-70 levels in normal subjects are biologically stable. ZAP-70 expression in CLL B-cells does not take into account the fact that ZAP-70 levels in T-cells can vary between patients. In their paper the authors describe a method when staining was performed in the absence (test) or in the presence of excess unconjugated SBZAP mAb (isoclonic control) to overcome the bias from enhanced ZAP-70 expression in residual T-cells in CLL. In their cohort of 32 patients with CLL and 10 normal controls the results of IGHV mutation status by ZAP-70 isoclonic and T/B ratios was similar.
Another way to obtain standardized results in ZAP-70 measurements is described in a publication by Preobrazhensky and coworkers published in this journal last year (5) where they report a novel way to use isotypic controls for flow cytometric analysis of ZAP-70 in CLL cells. A key feature is the use of isotypic controls at concentrations that differ from those recommended by the suppliers but selected to insure normal peripheral blood B-cells are ZAP-70 negative. They used isotypic control antibodies, where the concentrations were adjusted/optimized so that normal B-cells stained negatively for ZAP-70. An important finding of their study was that the distribution of ZAP-70 expression among the 70 investigated CLL patients was bimodal using this matched isotype cut-off method. It seems that the use of such experimentally optimized isotypic control method for assessing ZAP-70 expression in CLL cases has the potential to improve interpretation by placing the resultant ZAP-70 values in one of two groups and generating better correlation with IGHV mutational status compared with other methods. This method could also make interpretation of intermediate ZAP-70 values less subjective and possibly help reduce variation among different laboratories.
FLOW IN ALLERGY TESTING
Two activation markers have been described for more than a decade for basophil activation testing (BAT); the CD63 and CD203 measurements. The ectoenzyme CD203c (E-NPP3; pyrophosphatase/phosphodiesterase 3) was expressed on blood basophils, tissue mast cells, and their CD34+ progenitor cells but not on other blood leukocytes. In contrast to CD63, resting basophils show some degree of constitutive CD203c expression on their plasma membrane, whereas CD63 expression is closely related to basophil degranulation. In a publication of Sturm and coworkers in a previous issue of this journal (6) the authors defined the optimized conditions for CD203c-based BAT protocol and point out potential influencing factors. They found that longer periods of sample storage and the use of priming factors might confound BAT results based on CD203c determination. Thus they conclude that CD203c-based basophil activation test should be performed preferentially within 4 hour after taking the blood samples. Priming and degranulation-enhancing factors are not required for CD203c-based BAT. In contrast to skin testing, CD203c-based BAT can be performed also in patients undergoing anti-allergic treatment.
In the manuscript of Leysen et al. in this issue of the journal (7) the authors were the first to report on patients with immediate anaphylactic-like reactions to pholcodine – an opioid antitussive indicated for the treatment of unreproductive coughs - in which an IgE-mediated mechanism is suggested by quantification of sIgE and by two flow techniques the CD63-based BAT and the intracellular histamine concentration measurement (HistaFlow®). Briefly, in this technique intracellular histamine and its release is analyzed flow cytometrically by an enzyme affinity method using the histaminase diamine oxidase conjugated to fluorochromes. The authors suggest that the negative predictive value of the BAT/HistaFlow may be of help in excluding the diagnosis of immediate pholcodine allergy and also to further elucidate on the controversial putative cross-reactivity between pholcodine and neuromuscular blocking agents and other opioids.