The aim of the present study was to explore the diagnostic value of the immunophenotypic analysis of bone marrow mast cells (BMMC) in indolent systemic mast cell disease (SMCD) patients. For that purpose, a total of 10 SMCD patients and 19 healthy controls were analyzed. Our results show that BMMC from SMCD are different from normal BMMC with regard to both their light scatter and immunophenotypic characteristics. Accordingly, forward light scatter (FSC), side (90°) light scatter (SSC), and baseline autofluorescence levels were higher in BMMC from indolent SMCD patients than they were in control subjects. From the immunophenotypic point of view, the most striking findings were the constant expression of CD2 (P = .0001), CD25 (P = .0001), and CD35 (P = .06) molecules by BMMC from SMCD patients, markers that were absent from all normal controls. In contrast, CD71, absent in BMMC from indolent SMCD, was positive in BMMC from normal subjects. Although, slight differences between BMMC from SMCD patients and normal controls were found in several other markers, they did not reach statistical significance. In conclusion, our results show that simultaneous assessment of FSC/SSC and reactivity for the CD117, CD2, CD25, CD33, and CD35 forms the basis for the immunophenotypic characterization of BMMC from SMCD in adults and should be integrated with clinical and morphologic studies for the diagnosis of the disease.

MASTOCYTOSIS IS A rare disorder characterized by an increased number of tissue mast cells (MCs) that may involve either the skin alone or several organs such as bone marrow (BM), bone, spleen, liver, and lymph nodes. Among the systemic types, a recent consensus classification,1 which is based on the work of Travis et al,2 recognizes four different forms: (1) indolent systemic mast cell disease (SMCD), (2) aggressive mastocytosis (also known as lymphadenopathic mastocytosis with eosinophilia), (3) systemic mastocytosis associated with different hematologic disorders, and (4) mast cell leukemia.

The immunophenotype of normal human MCs has been extensively studied in different tissues.3-7 In addition, we have recently shown that bone marrow mast cells (BMMC) can be easily identified and enumerated using multiparametric flow cytometry on the basis of their strong reactivity for the CD117 and FcεRI antigens.8 In our experience,9 normal BMMC virtually always express CD9, CD11c, CD29, CD33, CD43, CD44, CD45, CD49d, CD49e, CD51, CD54, CD71, CD117, and FcεRI, while other markers such as CD11b, CD13, CD18, CD22, CD35, CD40, and CD61 displayed a variable expression. Apart from this report, the immunophenotype of BMMC has only been analyzed in samples from chronic myeloid leukemia,3,5 mast cell leukemia,10-12 and a case of malignant mastocytosis with circulating mast cells.13 Moreover, to the best of our knowledge, no extensive studies about the immunophenotype of BMMC have been performed in SMCD.

The aim of the present report is to define the immunophenotype of human BMMC from patients suffering from SMCD using a large panel of monoclonal antibodies (MoAbs) and to establish the possible role of immunophenotyping in the differential diagnosis between SMCD and normal/reactive mast cell. In addition, results on the reactivity obtained for each of the markers analyzed are expressed as molecules equivalent of soluble fluorochrome (MESF), which would allow the comparison with previous results obtained in BMMC from normal controls, as well as with future studies in which the same MoAb conjugates are used.

MATERIALS AND METHODS

A total of 12 BM samples were obtained from 10 patients suffering from indolent SMCD (six men and four women), with a mean age of 45 ± 14 years, ranging from 26 to 67 years. Cases of mastocytosis associated with hematologic disorders, aggressive mastocytosis, or mast cell leukemia were excluded from this study. The diagnosis of indolent SMCD was made according to the criteria proposed in previous reports.1,2,14-16 Table 1 shows the clinical and biological characteristics of each patient enrolled in the present study. Immunophenotypical studies were performed at diagnosis in eight cases (patients 1 to 7, and 9) and 1 and 8 years after the diagnosis in the other two cases (patients 10 and 8, respectively). At the moment of entering this study, five patients (2, 7, 8, 9, and 10) were under anti-H1 (chlorpheniramine maleate or hydroxyzine) and anti-H2 (ranitidine) therapy. Of these, three cases were receiving treatment with sodium cromoglycate (patients 7, 8, and 9) and one (patient 8) also received aspirin; no case was treated with interferon before entering this study. A total of 19 healthy volunteers undergoing either orthopedic surgery or BM harvest were sudied as normal controls.

Table 1.

Clinical and Biologic Characteristics of Patients With Indolent SMCD

Patient ID Age/Sex Tissues With MC InfiltrationPB BM Dysplasia % BMMC Tryptase-150Mediator-Related Symptoms Therapy-151
1  26/M  S, BM  N  Dyserithro  0.20  ND  No  None  
67/M  S, BM, B  N  No  0.42  4.2  Pruritus Anti-H1  
        Anti-H2  
30/M  S, BM  Eos  No  0.43  ND  No None  
4  37/F  S, BM  Eos  No  0.24 ND  No  None  
5  67/M  S, BM  Eos  No 0.36  ND  Pruritus  None  
6  51/F  S, BM N  No  0.015  ND  No  None  
7  51/F S, BM, GI, spleen-152 Eos, anemia-153 No  0.02  Pruritus, flush Syncope, abdominal pain Anti-H1 Anti-H2 CS  
     0.09 ND  No  Anti-H1  
        Anti-H2  
        CS  
8  49/M S, BM, B, GI, hepatomegaly  Eos  No  0.49  27 Pruritus, flush Syncope, abdominal pain Anti-H1 Anti-H2 CS Aspirin  
     0.29  6  Pruritus, flush abdominal pain Anti-H1 Anti-H2 CS Aspirin  
9  42/F  S, BM, B Eos  No  0.12  10  Pruritus, flush  Anti-H1 
        Anti-H2  
        CS  
10  33/M  S, BM, GI  N  No 0.15  ND  Pruritus  Anti-H1  
        Anti-H2 
Patient ID Age/Sex Tissues With MC InfiltrationPB BM Dysplasia % BMMC Tryptase-150Mediator-Related Symptoms Therapy-151
1  26/M  S, BM  N  Dyserithro  0.20  ND  No  None  
67/M  S, BM, B  N  No  0.42  4.2  Pruritus Anti-H1  
        Anti-H2  
30/M  S, BM  Eos  No  0.43  ND  No None  
4  37/F  S, BM  Eos  No  0.24 ND  No  None  
5  67/M  S, BM  Eos  No 0.36  ND  Pruritus  None  
6  51/F  S, BM N  No  0.015  ND  No  None  
7  51/F S, BM, GI, spleen-152 Eos, anemia-153 No  0.02  Pruritus, flush Syncope, abdominal pain Anti-H1 Anti-H2 CS  
     0.09 ND  No  Anti-H1  
        Anti-H2  
        CS  
8  49/M S, BM, B, GI, hepatomegaly  Eos  No  0.49  27 Pruritus, flush Syncope, abdominal pain Anti-H1 Anti-H2 CS Aspirin  
     0.29  6  Pruritus, flush abdominal pain Anti-H1 Anti-H2 CS Aspirin  
9  42/F  S, BM, B Eos  No  0.12  10  Pruritus, flush  Anti-H1 
        Anti-H2  
        CS  
10  33/M  S, BM, GI  N  No 0.15  ND  Pruritus  Anti-H1  
        Anti-H2 

Abbreviations: B, bone; BM, bone marrow; BMMC, bone marrow mast cells; GI, gastrointestinal tract; PB, peripheral blood; Eos, eosinophilia; ND, not detected; CS, Cromolyn sodium; N, normal; S, skin.

F0-150

Serum tryptase levels in UI/L.

F0-151

Therapy received at the moment entering the study.

F0-152

Splenomegaly detected by ultrasonography.

F0-153

Iron deficiency anemia.

In all cases, samples were collected in K3-EDTA anticoagulant and immediately diluted 1/1 (vol/vol) in phosphate-buffered saline (PBS). After collection, the sample was passed several times through a 25-mm gauge needle to disaggregate the BM particles and cell concentration was adjusted to 7.5 × 109 nucleated cells/L.

Immunologic marker analysis.

BM samples were analyzed by direct immunofluorescence using either triple or double-staining combinations of MoAb directly conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), and either the PE-cyanin 5 (PE-Cy5) fluorochrome tandem or PerCP. The following MoAb conjugates were used: (1) FITC-conjugated: CD2, CD10, CD13, CD14, CD15, CD16, CD19, CD20, CD22, CD25, CD33, CD34, CD44, CD71, (purchased from Becton Dickinson, San Jose, CA), CD38, CD65, (Caltag Laboratories, San Francisco, CA), CD11a, CD11b, CD11c, CD35, CD54, CD66 (CLB, Amsterdam, The Netherlands), CD29 (Coulter Corp, Miami, FL) CD21, CD30, CD42b, CD61 (Dakopatts, Copenhagen, Denmark), CD41a (Immunotech, Marseille, France), CD18, CD51 (Menarini Diagnostics, Barcelona, Spain), CD117 (Nichirei Corporation, Tokyo, Japan), CD9, CD43, CD49d, CD49e (Serotec, Oxford, UK) anti-IgE (The Binding Site, Birmingham, England), and BB4 (CD138) (Immunoquality Products, Gröningen, The Netherlands); (2) PE-conjugated: CD23 (purchased from Becton Dickinson), and CD117 (Nichirei Corporation); (3) PE/Cy5-conjugated: CD38, and HLA-DR (Caltag Laboratories); (4) Per-CP conjugated CD45 (Becton-Dickinson).

Briefly, 200 μL of the sample containing aproximately 1.5 × 106 nucleated cells were incubated for 10 minutes at room temperature with the above mentioned MoAbs. After this, erythrocytes were lysed by incubating cells for 10 minutes (room temperature) with 2 mL/tube of FACS lysing solution (Becton Dickinson) diluted 1/10 vol/vol in distilled water. Isotype-matched mouse nonspecific immunoglobulins and a tube stained for CD117-PE were used as negative and positive controls to assess nonspecific binding and BMMC autofluorescence, respectively.

Data acquisition was performed on a FACScan flow cytometer (Becton Dickinson) using the LYSYS II software program (Becton Dickinson). Initially, a minimum of 10,000 events/tube from the total BM cells was acquired. In addition, a second acquisition step, through a SSC/CD117 live gate, was performed to increase the sensitivity of the method for the analysis of MC present at a low frequency.8For data analysis, the Paint-A-Gate PRO software (Becton Dickinson) was used. The quantitation of positivity for each of the markers tested was performed using QuickCal beads (Flow Cytometry Standards Corporation, San Juan, Puerto Rico) and results were expressed as the mean number of molecules equivalent of soluble fluorochrome (MESF) obtained specifically for the MC. The threshold for positivity was 5,000 MESF for both FITC and PE after subtracting the autofluorescence levels obtained for BMMC.

Statistical methods.

Mean values and their standard deviations were calculated for all variables in each group of samples. The Mann-Whitney U and the χ2 tests were used to assess the statistical significance of the immunophenotypic differences observed between BMMC from SMCD patients and those of normal controls for continuous and dichotomic variables, respectively.

RESULTS

Despite their low frequency, MCs were clearly identified in all BM samples analyzed based on their strong CD117 expression (Fig 1). The mean number of BMMC in indolent SMCD patients was significantly higher (P > .001) than in normal controls (0.27% ± 0.19% and 0.021 ± 0.0025 of the nucleated BM cells analyzed, respectively). BMMC from indolent SMCD displayed a relatively homogenous FSC and SSC distribution, the values of both light scatter parameters being higher than those found for normal BMMC (Fig 2A and B). Moreover, the autofluorescence level was also higher in BMMC from indolent SMCD than it was in normal individuals (Fig 2C and D).

Fig. 1.

Representative dot plots of ungated (dot plots A to F) and CD117 gated events from a healthy control (left panel) and a patient suffering from indolent SMCD (right panel). Black dots correspond to BMMC. Total number of events in plots A to F is 10,000 (note that no mast cells are present among 10,000 ungated events in dot plots A, C, and E). The events shown in plots G to J correspond to the BMMC present in a total of 600,000 events analyzed. N, neutrophils; M, monocytes; L, lymphocyte; Er, erythroid nucleated cells.

Fig. 1.

Representative dot plots of ungated (dot plots A to F) and CD117 gated events from a healthy control (left panel) and a patient suffering from indolent SMCD (right panel). Black dots correspond to BMMC. Total number of events in plots A to F is 10,000 (note that no mast cells are present among 10,000 ungated events in dot plots A, C, and E). The events shown in plots G to J correspond to the BMMC present in a total of 600,000 events analyzed. N, neutrophils; M, monocytes; L, lymphocyte; Er, erythroid nucleated cells.

Fig. 2.

Representative example of the light scatter and immunophenotypic differences between BMMC (black dots) from healthy controls (left panel) and patients suffering from indolent SMCD. As can be seen, CD2 (E and F), CD25 (G and H), and CD35 molecules were positive in SMCD patients, but negative in normal controls. The events represented in each dot plot correspond to the CD117+gated cells present in a total of 600,000 events analyzed.

Fig. 2.

Representative example of the light scatter and immunophenotypic differences between BMMC (black dots) from healthy controls (left panel) and patients suffering from indolent SMCD. As can be seen, CD2 (E and F), CD25 (G and H), and CD35 molecules were positive in SMCD patients, but negative in normal controls. The events represented in each dot plot correspond to the CD117+gated cells present in a total of 600,000 events analyzed.

The immunophenotypical characteristics of BMMC from indolent SMCD are shown in Fig 2 and Table 2. As may be seen, three patterns of antigen expression were detected in BMMC from indolent SMCD patients: (1) markers that were constantly positive (CD2, CD9, CD11c, CD13, CD25, CD29, CD33, CD35, CD44, CD45, CD54, CD117, and FcεRI); (2) antigens that were always negative (CD10, CD11a, CD14, CD15, CD16, CD19, CD20, CD21, CD23, CD30, CD34, CD38, CD65, CD66, CD71, HLA-DR, and CD138); and (3) markers that were positive in a variable proportion of cases: CD11b (25%), CD18 (40%), CD22 (78%), CD41a (71%), CD42b (25%), CD43 (60%), CD49d (50%), CD49e (25%), and CD61 (28%). In addition, among those antigens that were positive, three groups could be identified according to the intensity of antigen expression detected: (1) strong positive markers (> 12,000 and > 50,000 MESF for FITC and PE, respectively): CD9, CD11c, CD25, CD33, CD44, CD49d, CD54, CD117 and FcεRI; (2) uniform dim positive antigens (from 5,000 to 12,000 MESF of FITC): CD2, CD11b, CD18, CD41a, CD49e, and CD51; and (3) antigens showing a variable intensity of expression (from 5,000 to 51,000 MESF of FITC): CD13, CD22, CD29, CD42b, CD43, and CD61.

Table 2.

Immunophenotypic Characteristics of BMMC in SMCD Patients and Healthy Controls

CD Indolent SMCD N = 10 Healthy Controls N = 19P Value
2* 9.87 ± 6.25  Negative .0001  
    5-24.9  
 100%  
826.30 ± 337.33  1,047.4 ± 474.5   NS 
  780.0-1,111.7   431.6-1692.0  
 100%  100% 
11b   6.5  6.25 ± 0.49  NS 
 <5-6.5  <5-6.6  
  25%   50%  
11c 20.1 ± 5.2   11.1 ± 2.2   NS 
 12.9-25.5  <5.0-14.2   
 100%   87%  
13 20.9 ± 21.5  13.8 ± 17.7  NS 
  7.0-56.9  <5.0-50    
 100%   77%  
18  5.8 ± 0.07  6.0 ± 0.1  NS  
 <5-5.9 <5.0-6.1    
  40%   62%  
22 11.9 ± 6.9   6.4 ± 1.0  NS 
  <5-25.5  <5.0-7.9    
  78%   50% 
25*  33.8 ± 16.57  Negative  .0001 
 18.1-48.5  
 100%  
29  15.0 ± 5.3  30.6 ± 10.1  .03  
  9.1-21.5  18.5-45.4 
 100  100  
33  58.9 ± 30.3 22.5 ± 14.0  .02  
  14.2-102.8   7.8-51.7 
 100%  100%  
35* 44.1 ± 0.14  Negative .006  
   44-44.2  
 100%  
41a*  7.9 ± 2.66  Negative  NS  
  <5-11.8 
  71%  
42b* 14.9  Negative  NS 
  <5-14.9  
  25%  
43   13.0 ± 10.96 26.6 ± 27.9  NS  
  <5-25.7  11.4-83.2 
  60%  100%  
44  140.8 ± 56.9  167.5 ± 99.6   NS  
  59.6-224.6  82.1-339.2  
 100%  100%  
45 100% 100%  NS  
49d  17.1 ± 2.6  21.2 ± 6.4   NS  
  <5-26.5  16.9-33.9 
  50%  100%  
49e   7.0  7.2 ± 1.7 NS  
 <5-7.0  5.0-9.4  
  25%  100% 
51   5.5  8.6 ± 3.4  NS  
 <5-5.5  5.6-13.2  
  20%  100%  
54  75.6 ± 55.8 14.2 ± 4.2   NS  
   6.5-134.6 <5.0-18.7   
 100%   75%  
61  11.5 ± 8.4  8.3 ± 2.5  NS  
  <5-17.5   <5-11.8 
  28%   85% 
71* Negative  6.9 ± 1.4 .004  
  5.0-8.3  
  100%  
117 174.1 ± 78.5   322.1 ± 192.2  .008 
  93.4-383.7   87.8-860.1  
 100%  100% 
FceRI  311.9 ± 273.7  428.3 ± 198.2 NS  
  41.3-747.5  288.1-568.5  
 100% 100%   
CD Indolent SMCD N = 10 Healthy Controls N = 19P Value
2* 9.87 ± 6.25  Negative .0001  
    5-24.9  
 100%  
826.30 ± 337.33  1,047.4 ± 474.5   NS 
  780.0-1,111.7   431.6-1692.0  
 100%  100% 
11b   6.5  6.25 ± 0.49  NS 
 <5-6.5  <5-6.6  
  25%   50%  
11c 20.1 ± 5.2   11.1 ± 2.2   NS 
 12.9-25.5  <5.0-14.2   
 100%   87%  
13 20.9 ± 21.5  13.8 ± 17.7  NS 
  7.0-56.9  <5.0-50    
 100%   77%  
18  5.8 ± 0.07  6.0 ± 0.1  NS  
 <5-5.9 <5.0-6.1    
  40%   62%  
22 11.9 ± 6.9   6.4 ± 1.0  NS 
  <5-25.5  <5.0-7.9    
  78%   50% 
25*  33.8 ± 16.57  Negative  .0001 
 18.1-48.5  
 100%  
29  15.0 ± 5.3  30.6 ± 10.1  .03  
  9.1-21.5  18.5-45.4 
 100  100  
33  58.9 ± 30.3 22.5 ± 14.0  .02  
  14.2-102.8   7.8-51.7 
 100%  100%  
35* 44.1 ± 0.14  Negative .006  
   44-44.2  
 100%  
41a*  7.9 ± 2.66  Negative  NS  
  <5-11.8 
  71%  
42b* 14.9  Negative  NS 
  <5-14.9  
  25%  
43   13.0 ± 10.96 26.6 ± 27.9  NS  
  <5-25.7  11.4-83.2 
  60%  100%  
44  140.8 ± 56.9  167.5 ± 99.6   NS  
  59.6-224.6  82.1-339.2  
 100%  100%  
45 100% 100%  NS  
49d  17.1 ± 2.6  21.2 ± 6.4   NS  
  <5-26.5  16.9-33.9 
  50%  100%  
49e   7.0  7.2 ± 1.7 NS  
 <5-7.0  5.0-9.4  
  25%  100% 
51   5.5  8.6 ± 3.4  NS  
 <5-5.5  5.6-13.2  
  20%  100%  
54  75.6 ± 55.8 14.2 ± 4.2   NS  
   6.5-134.6 <5.0-18.7   
 100%   75%  
61  11.5 ± 8.4  8.3 ± 2.5  NS  
  <5-17.5   <5-11.8 
  28%   85% 
71* Negative  6.9 ± 1.4 .004  
  5.0-8.3  
  100%  
117 174.1 ± 78.5   322.1 ± 192.2  .008 
  93.4-383.7   87.8-860.1  
 100%  100% 
FceRI  311.9 ± 273.7  428.3 ± 198.2 NS  
  41.3-747.5  288.1-568.5  
 100% 100%   

Results expressed as mean ± SD and range of antigen expression in MESF (units × 103), and percentage of positive cases. P values were calculated for differences in the intensity of antigen expression and proportion of positive cases. CD10, CD11a, CD14, CD15, CD16, CD19, CD20, CD21, CD23, CD30, CD34, CD38, CD65, CD66, CD138, and HLA-DR were negative in BMMC from all SMCD and controls studied.

Abbreviations: NS, statistically not significant; ND, not determined.

*

Proportion of positive cases.

PE/Cy5 conjugated MoAbs.

PE-conjugated MoAbs.

After comparing the immunophenotype of BMMC from indolent SMCD patients and healthy subjects, significant differences were observed regarding both the incidence of positivity and the fluorescence intensity (Table2). Accordingly, in all indolent SMCD patients, BMMC expressed the CD2, CD25, and CD35 antigens, which were never found in BMMC from normal controls (P = .0001, .0001, and .006, respectively). In contrast, the CD71 molecule was constantly present in normal controls, but never detected in indolent SMCD cases (P = .004). Other molecules such as CD41a and CD42b were present in a variable proportion of SMCD cases, but constantly absent in normal controls (P = .6 and .06, respectively). Additional differences between BMMC from indolent SMCD cases and healthy individuals were observed upon analyzing the levels of mean fluorescence intensity estimated for several antigens: CD33 expression was significantly higher in SMCD patients (P = .02), while CD29 and CD117 expression was greater in healthy subjects (P = .03 and .008, respectively).

DISCUSSION

The present report represents a first attempt at the extensive characterization of the immunophenotype of BMMC from indolent SMCD adult patients using a large panel of MoAb. Our major goal was to explore the use of immunophenotyping for the differential diagnosis between SMCD and normal/reactive MC. Although, as expected, the mean number of BMMC in indolent SMCD was significantly higher than in normal controls,8 the overall number of BMMC in indolent SMCD was low. This is concordant with histologic studies that show a low number of MC17,18 with focal distribution.2,17,19-23 

Interestingly, BMMC from SMCD displayed light scatter characteristics that were higher than those found for their normal counterpart, as well as a greater level of autofluorescence. Light scatter properties of cells analyzed at flow cytometry to a large extent reflect morphologically-related features such as cell size and internal complexity.24 As a matter of fact, a slightly larger size has been reported for BMMC in SMCD25 and this could explain the higher FSC values found in the present study. Interestingly, both piecemeal and anaphylactic degranulation have been described in vivo at the ultrastructural level in MC from different tissues,26-30 including BMMC31 from SMCD patients. Accordingly, MC undergoing degranulation display several changes regarding granule size, shape, and contents,29,30,32 and these morphologic changes, together with an increased content in endoplasmic membranes, Golgi apparatus and endoplasmic reticulum, may explain the increased SSC (internal complexity) of BMMC from SMCD found in the present study. Additionally, this would also help to increase the autofluorescence levels of SMCD BMMC. Isolation of mast cells from a SMCD using a fluorescence activated cell sorter according to previously described methods8 confirmed that these MC displayed an abnormal morphology (data not shown).

Increased autofluorescence of SMCD BMMC may limit the measurement of the fluorescence levels obtained for specific antigens detected through the use of immunofluorescence techniques. Therefore, the assessment of the immunophenotype of BMMC from SMCD patients should take into account the autofluorescence levels of these cells, and the fluorescence intensity for a specific marker should be calculated after subtracting the baseline autofluorescence of the cells under study. To allow the comparison of the results of the present report with those of other studies in which the same MoAb conjugates are used, results on the reactivity obtained for each of the markers analyzed were expressed as MESF after subtracting the MESF values corresponding to the mean autofluorescence level obtained for those specific mast cells.

From the immunophenotypic point of view, the most striking findings were the expression of CD2, CD25, and CD35 molecules by BMMC from all SMCD patients, markers that were constantly absent in normal BMMC.3,5,9,33,34 Coexpression of both CD2 and CD25 by BMMC could be considered as characteristic of indolent SMCD, but not normal and reactive BMMC,9 and it could be of great help for the differential diagnosis between SMCD and reactive mastocytosis. Nevertheless, reactivity for CD35 cannot be pathognomonic of SMCD, because although negative in MC from several tissues5,7,34-36 including normal BM,9 it has been observed to be weakly positive in BMMC from some patients (27%) with B-cell malignancies.9 On the other hand, the reactivity for CD71 (transferrin receptor) was shown to be constantly negative in indolent SMCD cases, while positive in BMMC from normal subjects,9 as well as in the MC from a case of mast cell leukemia.12 However, because CD71 expression in normal BMMC was always weak (from 5 to 6.5 × 103 MESF), this would limit its use in the differential diagnosis of SMCD. It should be noted that this antigen is absent in human MC from different tissues.3,5,35 

The CD29 and the myeloid-associated markers CD33 and CD117, although present in BMMC from all indolent SMCD patients and normal controls studied, displayed a significantly different intensity of expression in both groups of individuals. In this sense, while reactivity for CD33 was higher in SMCD patients, CD29 and CD117 were expressed at a greater intensity in BMMC from control subjects.

In summary, our results show that multiparametric flow cytometry using direct immunofluorescence on erythrocyte-lysed whole BM is of great help for the diagnosis of adult indolent SMCD and its differential diagnosis with reactive mastocytosis. Simultaneous assessment of FSC/SSC and reactivity for the CD117, CD2, CD25, CD29, and CD33, in the presence of BM involvement, forms the basis for the immunophenotypic diagnosis of SMCD in adults.

ACKNOWLEDGMENT

We thank Professor Frank K. Austen for reading the manuscript.

Supported by Grant No. FIS 95/0768 from the Fondo de Investigaciones Sanitarias de la Seguridad Social (to B.D.-A.).

Address reprint requests to Alberto Orfao, MD, Servicio Central de Citometrı́a, Hospital Clı́nico Universitario, Paseo de San Vicente s/n, 37007 Salamanca, Spain.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

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