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Systemic mast cell disease is characterised by abnormal proliferation and accumulation of mast cells in bone marrow or other non-cutaneous organs.1 The abnormal proliferation of mast cells is also associated with other haematological disorders, classified as systemic mastocytosis with associated clonal haematological non-mast-cell lineage disease (SM-AHNMD). However, the presence and dysplasia of mast cells can easily be overlooked during routine bone marrow examination. Therefore, the laboratory staff and oncologists in our hospital decided to specifically stain mast cells with toluidine blue in bone marrow aspirate specimens of patients suspected to have different types of systemic mastocytosis (SM), or other clonal haematological disorders, such as myelodysplastic syndromes (MDS), thereby reducing the chance of missing the increased presence and dysplasia of mast cells in bone marrow specimens.2
MDS are classified by the WHO as myeloid neoplasms, and are characterised by cytopenia and dysplasia in one or more myeloid cell lines.3 This is a result of ineffective haematopoiesis and leads to increased risk of development of acute myeloid leukaemia. To establish the diagnosis of MDS can be challenging, particularly for low-grade MDS.4 Therefore, new diagnostic tools—for example, flow cytometry, to facilitate low-grade MDS diagnosis are needed.
In our laboratory, since the introduction of toluidine blue staining of bone marrow specimens, we have frequently found an increased number of mast cells, often showing dysplasia, in bone marrow aspirates of patients suspected to have MDS. To investigate whether this finding might be helpful in the diagnosis of MDS, we analysed whether the number and dysplasia of mast cells were indeed increased in patients diagnosed with MDS. Interestingly, we found that mast cell numbers and dysplasia are significantly increased in patients diagnosed with low-grade MDS.
Eighty-five routine bone marrow aspirates were evaluated at our clinical laboratory by two experienced technicians. The age of the patients ranged from 37 to 91 years, with a median of 66 years. The final diagnosis, based on WHO criteria, was made by their physician. Both the technician and the physician had no knowledge of the conclusion of the diagnostic process or the mast cell count, respectively. Depending on the diagnosis, these cases were divided into four groups—A: no abnormalities (n=27), B: low-grade MDS (not refractory anaemia with excess blasts (RAEB)-1 and RAEB-2) (n=9), C: benign abnormalities (total: n=19, divided into iron deficiency (n=13) and other benign abnormalities (n=6)), D: haematological malignancies (total: n=29, divided into myeloid neoplasms (n=15; chronic myeloid leukaemia, n=2; acute myeloid leukaemia, n=9; essential thrombocytosis, n=2; polycythaemia vera, n=2) and lymphoid neoplasms (n=84)). In addition, two samples with SM were evaluated as positive controls. All samples were stained with May–Grünwald Giemsa and toluidine blue, the latter for the identification of mast cells (figure 1).
The number of mast cells and mast cell dysplasia (spindle-shaped cells and/or hypogranular cells, reported as percentage of total mast cells) were quantified in the bone marrow specimens by evaluating at least 10 spicules. The data used for statistical analysis are based on the mean value of mast cell number per spicule and percentage of dysplastic mast cells for each bone marrow aspirate, as observed by both technicians. Statistical analysis was performed with SPSS V.19 software (SPSS Inc, Chicago, Illinois, USA). The data were analysed with the Kruskal–Wallis test, followed by a pairwise multiple comparison (Dunn's method); p<0.05 was considered significant.
All bone marrow aspirates were evaluable and included in our analysis. Quantification of the mast cells showed significantly higher numbers of mast cells in the bone marrow aspirates of patients diagnosed with MDS (Kruskal–Wallis test: p<0,01, figure 2A). Dunn's pairwise comparison shows significant differences between mast cell count in the MDS group and in the control group or the group with haematological malignancies. The difference in the number of mast cells remained significant when the MDS group was compared with the group with myeloid malignancies only (exclusion of lymphoid malignancies, data not shown). In addition, dysplasia of mast cells was more prominent in the MDS group (p<0.05) (figure 2B). Samples from patients with SM were evaluated as a positive control and showed increased number (>200 mast cells per spicule, data not shown) and dysplasia of mast cells in the bone marrow. Receiver operating characteristic analysis examining mast cell count for the diagnosis of MDS in this patient population yielded an area under the curve for mast cell count of 0.76 (p<0,01, figure 3).
In this study, we described the added value of toluidine blue staining of bone marrow aspirates and concomitant mast cell count for the diagnosis of low-grade MDS. To establish the diagnosis of MDS, particularly low-grade MDS, can be challenging, since dysplasia is a subjective and qualitative judgment. Our results suggest that adding toluidine blue staining and mast cell count to the standard laboratory routine may facilitate MDS diagnosis. In contrast to techniques which have been evaluated for the diagnosis of MDS, such as flow cytometry, this approach is easier, cheap and provides an objective, quantitative result.
The number of mast cells is significantly increased in bone marrow aspirates of patients with MDS. Moreover, mast cells in patients with MDS display a more dysplastic phenotype. These patients cannot be diagnosed as having SM-AHNMD, since the total quantity and percentage dysplasia of mast cells do not reach the criteria for SM-AHNMD, which are multifocal, dense infiltrates of mast cells (≥15 mast cells in aggregates, major criterion), and, as a one of the minor criteria, >25% dysplasia of mast cells in biopsy sections of bone marrow or other extracutaneous organs.3
In our patient group clinical features of MDS were most prominent, therefore data for the other minor criteria (KIT mutation, aberrant CD2 and/or CD25 expression of mast cells and serum tryptase >20 ng/mL) were not available. From experience, it is known that most SM-AHNMD bone marrow aspirates have some degree of minor morphological dysplasia, which may not be diagnostic of MDS.5 Possibly, a number of patients have an overlapping syndrome between MDS and SM-AHNMD. The analysis of a toluidine blue staining might be useful in the identification of this variant; however, further study is needed to support these hypotheses.
The evaluation of mast cells in patients with MDS has been described previously by Dunphy,6 who found an increased level of mast cells in 16% of bone marrow from patients with MDS, whereas in patients with myeloproliferative disorders an increased level of mast cells was found in up to 70% of bone marrow aspirates. In contrast, our results point towards higher number of mast cells in MDS as compared with other haematological malignancies, including six cases of myeloproliferative disorders. This discrepancy might be due to the difference in methods used for the detection of mast cells in the bone marrow aspirates. We performed a toluidine blue staining on the bone marrow samples, which greatly facilitates the detection of mast cells in the bone marrow spicules. Therefore, we were able to quantify the number of mast cells, rather than using a more qualitative measurement as described by Dunphy.6 In addition, Dunphy showed that the presence of mast cells is specifically increased in patients with chronic myeloid leukaemia, which was only diagnosed in two patients from our study group.
Our data suggest that mast cell presence and dysplasia are significantly increased in bone marrow of patients with low-grade MDS, in comparison with other haematological malignancies. These findings need to be confirmed in a large, prospective study and may help to facilitate the diagnosis of MDS.
MWMS and KC contributed equally.
Contributors PK designed the experiment, KC and PdW performed the analysis, MWMS and KC wrote the article.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.