Basophilia is an important feature of MPD, often a diagnostic finding, and sometimes even of prognostic significance.20,22,23,24,25,26,27,28
However, so far no reliable immunohistochemical marker for basophil detection and enumeration in MPD has become available. We have established 2D7 as a powerful new marker for the immunohistochemical analysis of basophils in patients with MPD. The results of our study show that 2D7 is specific for basophils in normal bone marrow as well as bone marrow in patients with MPD, and that the numbers of basophils can easily be quantified (or estimated) in 2D7 stained bone marrow sections. The 2D7 antibody should therefore be considered a novel helpful marker for basophil detection in haematopathology.
Although the 2D7 antibody is known to label basophils specifically in inflamed tissues41,42,43
and in normal peripheral blood,33
it was of pivotal importance to reconfirm the specificity of 2D7 for basophils in patients with MPD. To address this issue, two different approaches were used. First, purified CML basophils and CML cells depleted of basophils, were analysed. In these experiments, we were able to show that 2D7 is selectively expressed in CML basophils, but is not expressed in basophil depleted cell fractions. In consecutive experiments, serial section staining was carried out in order to reconfirm that the 2D7 stained bone marrow cells express the established phenotype of human basophils.14,15,16
Various previous studies have shown that basophils express a unique cell surface marker profile, including myeloid determinants as well as activation linked cell surface antigens.10,11,14,15,16,34,44
This phenotype has been described for normal blood basophils and for CML derived basophils.10,14,15,16
However, no systematic immunohistochemical analysis of bone marrow basophils has been presented so far. We now show that 2D7+
basophils in the bone marrow in patients with CML express a unique phenotype which corresponds largely to the cell surface phenotype of (normal and CML) basophils. However, some of the antigens detected in CML basophils by immunohistochemistry, like CD4 or CD15, are usually not detectable on the surface of normal blood basophils.10,14,15,16,34
With regard to CD15, this antigen is indeed expressed on the surface of basophils, but is masked by gangliosides and is thus detectable only after treatment with neuraminidase.10
In the case of CD4, the discrepancy may have several explanations. First, the antibody used may cross react with other antigens or may show non‐specific binding to basophils. An alternative explanation may be that CML basophils express very small amounts of CD4 on their cell surface. In this regard it is noteworthy that CML basophils can react with CD4 antibodies in cell surface staining experiments when these cells are kept in culture for several days.10
Whether CML basophils indeed express functional CD4 and thereby can interact with class II HLA antigen or the HIV virus remains presently unknown.
In CML, the numbers of basophils are almost invariably increased and correspond to the phase of disease.20,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45
Especially in the accelerated phase of CML, many patients present with highly upregulated numbers of basophils.22,23,24,25,26,45
This phenomenon was confirmed in the present study by 2D7 staining. In fact, the largest numbers of 2D7+
cells were recorded in patients with accelerated phase CML. Thus the 2D7 antibody is a reliable marker and helpful to enumerate basophil counts in various phases of CML. In this regard, it is noteworthy that disease acceleration in CML is even defined by bone marrow basophilia.28,35,36
In addition, 2D7 may be helpful for monitoring of basophils—that is, their decrease during the first months of treatment with tyrosine kinase inhibitors.
In a few CML patients, basophilia is excessive and results in the clinical (pathological) picture of (secondary) basophilic leukaemia.37,38
Such excessive basophilia was also found in one of the patients examined in the current study. In this particular patient, more than 30% of all nucleated bone marrow cells appeared to react with the 2D7 monoclonal antibody. Thus the 2D7 antibody may also be a helpful marker to confirm the presence of basophilic leukaemia.
Basophils are increased not only in CML, but also in most other subtypes of MPD.19,21,22
Therefore we extended our analyses using 2D7 monoclonal antibody to various groups of patients with MPD. However, interestingly, the numbers of 2D7+
bone marrow basophils were found to be in the same range in patients with other MPD compared with normal/reactive bone marrow. An interesting finding was that bone marrow basophils are not increased in patients with indolent systemic mastocytosis. In these patients, the mast cell infiltrates were clearly composed of 2D7–
cells, and the remaining normal appearing marrow did not contain increased numbers of 2D7+
cells when compared with normal/reactive bone marrow. This observation provides further evidence that basophils and mast cells represent two different haematopoietic cell lineages.15
No evidence for the previously reported basophil/mast cell hybrids46
was found in the current study, in agreement with the observations of Foster et al
An important question addressed in our study was whether the numbers of 2D7+ bone marrow cells correlate with other basophil related or disease related indices. Indeed, the numbers of bone marrow basophils did correlate with the numbers (percentages) of peripheral blood basophils, histamine levels, and to a lesser degree, the leucocyte counts. These findings further document the value of the 2D7 monoclonal antibody in the immunohistochemical quantification of basophilia in patients with MPD. On the other hand, we quantified 2D7+ cells on a “per mm2” basis, so a possible influence of variation in cellularity between patients and disease categories needs to be taken into account. However, the cellularity among MPD patients did not differ extensively, so that such an influence may not change the overall message of this report. In this regard it should also be pointed out that in the non‐CML MPDs, the numbers of 2D7+ cells did not vary significantly from each other and not much from controls, so that the stain may not be helpful in separating non‐CML MPD from reactive basophilia.
In summary, we have established a novel immunohistochemical staining technique for the detection and enumeration of basophils in routinely processed bone marrow trephine biopsy sections. This may be a helpful new approach in haematopathology.