CME/SAM. Olga Pozdnyakova, MD, PhD, 1 Svetlana Kondtratiev, MD, 1,2 Betty Li, MS, 1 Karry Charest, 1 and David M. Dorfman, MD, PhD 1.

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1 Hematopathology / New Mastocytosis Flow Cytometry Approach High-Sensitivity Flow Cytometric Analysis for the Evaluation of Systemic Mastocytosis Including the Identification of a New Flow Cytometric Criterion for Bone Marrow Involvement Olga Pozdnyakova, MD, PhD, 1 Svetlana Kondtratiev, MD, 1,2 Betty Li, MS, 1 Karry Charest, 1 and David M. Dorfman, MD, PhD 1 Key Words: Systemic mastocytosis; Flow cytometry; CD2; CD25; Discrete mast cell population CME/SAM DOI: /AJCP5PJWK4QFHWHM Upon completion of this activity you will be able to: define 8 World Health Organization criteria used for the diagnosis of systemic mastocytosis and other mast cell neoplasms. list aberrant immunophenotypic markers present on the neoplastic mast cells and discuss their frequency and utility. apply optimal reagents and gating strategy in flow cytometric analysis of cases of known or suspected mast cell neoplasm. Abstract We used high-sensitivity flow cytometry to assess 93 bone marrow aspirates for involvement by systemic mastocytosis. Aberrant CD2/CD25 expression by CD117-gated mast cells was seen in 34 samples (37%), with the majority of mast cells expressing both markers (n = 23; 68%). In 24 cases, a discrete population of mast cells within the CD117-bright gate correlated with a positive morphologic finding in the biopsy, even in the absence of an aberrant immunophenotype. A discrete CD117-bright population, when considered a positive criterion, increases analytic sensitivity from 77% to 95%, exceeding the sensitivity of morphologic analysis (69% for aspirate and 85% for biopsy). We conclude that flow cytometry is a sensitive and specific test for the presence of systemic mastocytosis, particularly when the presence of a discrete CD117-positive mast cell population is regarded as a diagnostic criterion. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 480. Exam is located at Systemic mastocytosis is a rare myeloproliferative neoplasm that is characterized by an increased number of neoplastic mast cells in the extracutaneous organs with the bone marrow being the most frequent site of involvement. The 8 World Health Organization (WHO) Classification of Tumours of Haematopoietic and Lymphoid Tissues recognizes 6 variants of systemic mastocytosis: indolent systemic mastocytosis, systemic mastocytosis with associated clonal hematologic non mast cell lineage disease, aggressive systemic mastocytosis, mast cell leukemia, mast cell sarcoma, and extracutaneous mastocytoma. 1 For many years, morphologic evaluation has been considered the gold standard in diagnosing systemic mastocytosis, and the presence of multifocal aggregates of more than 15 mast cells has become the only major criterion for diagnosing systemic disease. 2,3 However, in some instances, mast cell aggregates are absent or small. In these cases, the diagnosis of systemic mastocytosis relies on the presence of at least 3 of 4 minor criteria, which include abnormal mast cell morphology, aberrant immunophenotypic expression of CD2 and/or CD25 by the neoplastic mast cells, an activating KIT D816V point mutation, and a serum tryptase level higher than 20 ng/ml. 1,2,4,5 Early flow cytometric studies by Escribano et al 5 showed that neoplastic mast cells share some immunophenotypic features with normal/reactive mast cells, including high-level expression of CD117, which has been used as a gating strategy. The same flow cytometric studies have consistently detected aberrant expression of CD2, a cell-adhesion molecule present on T cells, and CD25, the alpha chain 416 Am J Clin Pathol 2012;138: DOI: /AJCP5PJWK4QFHWHM

2 Hematopathology / Original Article of the interleukin (IL) 2 receptor that is present on activated B and T cells, on all mast cells obtained from cases of indolent systemic mastocytosis. 6,7 However, Morgado et al 8 questioned the usefulness of CD2 as a useful marker for the diagnosing of systemic mastocytosis, because its expression as a sole marker of mastocytosis was seen only in a small subset of cases. Although the diagnostic value of CD25 expression has been shown to exceed that of CD2, studies have demonstrated aberrant CD25 expression in approximately 80% of systemic mastocytosis cases, at a much lower frequency than was originally detected. 9 These discrepancies in the aberrant immunophenotypic expression on neoplastic mast cells may be explained by patient selection bias, small patient cohorts, and nonstandardized flow cytometric techniques. At the Brigham and Women s Hospital (BWH) Hematology Laboratory (Boston, MA) we used a uniform highsensitivity flow cytometric approach for the evaluation of systemic mastocytosis that includes collecting a large number of events (at least 1,000,000 events whenever possible). Here we report our findings from the analysis of 93 bone marrow samples from patients with known or suspected systemic mastocytosis. Gating for mast cells was performed based on the side scatter properties and CD117- bright expression with subsequent evaluation for CD2 and/ or CD25 aberrant expression. The flow cytometric results were compared with the bone marrow biopsy, clinical, and other laboratory findings. In addition to comparing the specificity and sensitivity of this flow cytometric approach with those of other diagnostic modalities and reporting the frequency of an aberrant immunophenotype in cases of systemic mastocytosis, we identified a new flow cytometric criterion for diagnosing bone marrow involvement by a mast cell disorder. Materials and Methods Patients In the period between July 9 and December 2011, BWH Hematology Laboratory performed flow cytometric analysis on 93 consecutive bone marrow aspirate samples from 78 patients submitted for evaluation for systemic mastocytosis. The patient characteristics and clinical diagnoses are presented in Table 1. Flow Cytometric Analysis for Systemic Mastocytosis Flow cytometric analysis for systemic mastocytosis was performed using a Becton-Dickinson FACSCanto flow cytometer (BD Biosciences, San Jose, CA) using CD117, CD2, and CD25 antibodies with their respective Table 1 Patient Demographics Characteristics No. * Patients 78 Bone marrow samples 93 Age, y Range Median 51 Sex, M/F ratio 0.5 Clinical diagnosis Systemic mastocytosis 23 Indolent 19 Aggressive 2 SM with AHNMD 2 Mast cell sarcoma 1 Cutaneous mastocytosis 4 Monoclonal mast cell activation syndrome 8 Mast cell activation syndrome 18 Anaphylaxis 16 Angioedema 2 Chronic urticaria 2 No clinical information 4 SM with AHNMD, systemic mastocytosis with associated hematologic non mast cell disorder. * No. of patients unless otherwise indicated. fluorochrome isotype controls (BD Biosciences). At least 1,000,000 events were collected for analysis whenever possible. In cases with limited cell counts the entire specimen was analyzed. Number of collected events ranged from,000 to 3,,000 events. The gating was performed on CD117-bright cells with variable side scatter mast cell gate with the number of events ranging from 23 to 102,794. Aberrant CD2 and/or CD25 expression was analyzed within the CD117-positive mast cell gate and the results were compared with the isotype controls. The results were interpreted as positive when a discrete population of CD2- and/or CD25-expressing cells was seen. The results were interpreted as suspicious when an increased number of CD2- and/or CD25-positive events was seen compared with the isotype control without a discrete positive population. The results were interpreted as negative when no difference was seen in CD2 and/or CD25 expression compared with the isotype control Image 1. Comparison of Flow Cytometric Results With Other Pathologic and Clinical Findings Flow cytometric results from the bone marrow aspirates submitted for systemic mastocytosis evaluation were compared with the results of the concurrent bone marrow biopsy and aspirate findings. Only 1 case did not have an aspirate available for morphologic examination. Morphologic and immunophenotypic results were correlated with other clinical and laboratory data, including serum tryptase level, molecular diagnostic assessment for the KIT D816V Am J Clin Pathol 2012;138: DOI: /AJCP5PJWK4QFHWHM 417

3 Pozdnyakova et al / New Mastocytosis Flow Cytometry Approach mutation, and clinical diagnoses. Clinical information was collected through chart review, and the diagnosis of systemic mastocytosis was made using the 8 WHO criteria of either 1 major and 1 minor criterion or 3 minor criteria. Statistical Analysis Sensitivity and specificity were analyzed using the Richard Lowry clinical calculator (Vassar College, Poughkeepsie, NY). 10 For sensitivity and specificity analyses, flow cytometric, biopsy, and aspirate findings interpreted as suspicious for involvement by systemic mastocytosis were included with the positive for systemic mastocytosis cases. Results Flow Cytometric Assessment of Bone Marrow Involvement by Neoplastic Mast Cells Based on Aberrant CD2 and/or CD25 Expression and Correlation With Morphologic Findings We evaluated 93 consecutive bone marrow samples from 78 patients using a high-sensitivity flow cytometric approach for the diagnosis of systemic mastocytosis. Five patients with systemic mastocytosis had sequential follow-up evaluations. The flow cytometric analysis yielded a positive result in 19 samples (20%; Image 1A) and a suspicious result in 15 samples (16%; Image 1B), for a total of 34 A B C 2 1 Image 1 Examples of positive (A), suspicious (B), and negative (C) flow cytometric analyses based on aberrant CD2 and/or CD25 expression on mast cells. 418 Am J Clin Pathol 2012;138: DOI: /AJCP5PJWK4QFHWHM

4 Hematopathology / Original Article samples (37%), and a negative result in 59 samples (64%; Image 1C). In the majority of cases, the neoplastic mast cells demonstrated aberrant expression of both CD2 and CD25 (n = 23; 68%). Aberrant expression of CD25 only was seen in 9 cases (26%) and expression of CD2 as the sole marker was seen in 2 cases (6%). Table 2 shows the correlation of flow cytometric results with morphologic findings from the bone marrow biopsy and aspirate smear. An example of a positive case of systemic mastocytosis that includes bone marrow biopsy and flow cytometric findings is presented in Image 2. Evaluation of bone marrow biopsy findings demonstrated 28 cases positive for systemic mastocytosis (30%) with 8 cases that were interpreted as suspicious (9%), for a total of 36 cases (39%). Morphologic assessment of the bone marrow aspirate identified 18 positive cases (20%) and 11 suspicious cases (12%), for a total of 29 cases (32%). All positive aspirate cases had positive biopsy findings. All 3 modalities demonstrated a similar number of cases negative for systemic mastocytosis: 64% for flow cytometry (n = 59), 61% for biopsy (n = 57), and 68% for aspirate smear (n = 63). A Table 2 Assessment of Bone Marrow Involvement by Neoplastic Mast Cells Using Immunophenotypic and Morphologic Methods (n = 93) * Flow Cytometry Standard Revised Biopsy Aspirate Positive 19 (20) 29 (31) 28 (30) 18 (20) Suspicious 15 (16) 12 (13) 8 (9) 11 (12) Negative 59 (64) 52 (56) 57 (61) 63 (68) * Data are given as number (percent). In one case, aspirate smear analysis was not performed. In addition, we performed flow cytometric analysis on 10 cases of Waldenström macroglobulinemia (lymphoplasmacytic lymphoma) that may show increased numbers of mast cells as a response to the neoplastic lymphocytes. None of the cases showed aberrant expression of CD2 and/or CD25 by the CD117-bright mast cells (results not shown). B C 2 1 D CD2 Image 2 Example of a case of systemic mastocytosis. A, bone marrow biopsy specimen (H&E, 600) with sheets of mast cells. B, Mast cell tryptase immunoperoxidase stain that is positive in the neoplastic cells ( 600). C, Flow cytometric analysis with a discrete population of CD117-bright mast cells. D, The mast cells aberrantly express CD25 on flow cytometry. CD117 CD25 Am J Clin Pathol 2012;138: DOI: /AJCP5PJWK4QFHWHM 419

5 Pozdnyakova et al / New Mastocytosis Flow Cytometry Approach Table 3 Correlation of Flow Cytometry Results With Other Concurrent Results * Other Results Elevated Tryptase KIT D816V Systemic Flow Cytometry Results Biopsy Levels (>20 ng/ml) Mutation Mastocytosis Positive for mast cell neoplasm (n = 34) Positive Negative Not available Negative for mast cell neoplasm (n = 59) Positive Negative Not available * Flow cytometry results are based on aberrant CD2 and/or CD25 expression. Other concurrent results include bone marrow biopsy findings, tryptase levels, KIT D816V mutation status, and clinical diagnosis of systemic mastocytosis. Data are given as numbers of cases. One patient with diagnosis of mast cell sarcoma carried codon 419 mutation. Table 4 Flow Cytometric Results for Patients With Clinical Diagnosis of Systemic Mastocytosis (n = 39) FC Aberrant Discrete Population BM Result Markers of Mast Cells Biopsy 1 pos CD25 Present pos 2 pos CD25 Present pos 3 pos CD25 Present pos 4 pos CD25 Present pos 5 pos CD25 Present pos 6 sus CD25 Present neg 7 pos CD25 and CD2 Absent pos 8 pos CD25 and CD2 Absent pos 9 sus CD25 and CD2 Absent sus 10 neg Absent neg 11 sus CD25 and CD2 Absent neg 12 sus CD25 and CD2 Absent neg 13 pos CD25 Present pos 14 pos CD25 and CD2 Absent sus 15 sus CD25 and CD2 Absent neg 16 sus CD25 and CD2 Absent pos 17 pos CD25 and CD2 Present pos 18 pos CD25 and CD2 Present pos 19 pos CD25 and CD2 Present pos 20 neg Present pos 21 sus CD25 and CD2 Absent pos 22 sus CD25 and CD2 Present sus 23 neg Present pos 24 neg Present pos 25 neg Present pos 26 neg Present pos 27 neg Present pos 28 neg Present pos 29 neg Present pos 30 neg Absent sus 31 sus CD25 Absent sus 32 pos CD25 and CD2 Present pos 33 pos CD25 and CD2 Absent pos 34 pos CD25 and CD2 Present pos 35 pos CD25 and CD2 Present pos 36 pos CD25 and CD2 Present neg 37 sus CD25 and CD2 Absent sus 38 sus CD25 and CD2 Absent pos 39 pos Present pos BM, bone marrow; FC, flow cytometry; neg, negative; pos, positive; sus, suspicious. Correlation of Flow Cytometric Results Based on Aberrant CD2 and/or CD25 Expression With Bone Marrow Biopsy, Laboratory, and Clinical Data Initial flow cytometric analysis for mast cell involvement was performed based on the identification of aberrant CD2 and/or CD25 expression by the CD117-bright gated mast cells. Results that were positive or suspicious for involvement by a mast cell neoplasm in both the flow cytometric and biopsy assessments were combined into 1 group. Flow cytometry results that were positive/suspicious (n = 34) and negative (n = 59) for mast cell neoplasm were correlated with the biopsy findings, tryptase levels, KIT mutation status, and the overall clinical diagnosis of systemic mastocytosis Table 3. In the positive/suspicious flow cytometry group, the correlation with other findings supporting a diagnosis of systemic mastocytosis was good: the biopsy findings were positive in 27 of 34 cases, an elevated serum tryptase level was seen in 23 of 34 cases, and a KIT mutation was detected in 26 of 34 cases. These results led to the diagnosis of systemic mastocytosis in 30 of 34 cases. One of the patients in this group had a diagnosis of mast cell sarcoma involving the bone marrow and this patient was included with other cases of systemic mastocytosis. In 2 cases in the positive/suspicious flow cytometry group, the criteria for the diagnosis of systemic mastocytosis were not fulfilled. In the negative flow cytometry group, the majority of the biopsy and clinical findings did not support the diagnosis of systemic mastocytosis: the biopsy results were negative in of 59 cases, tryptase levels were less than 20 ng/ml (20 µg/l) in 40 of 59 cases, and KIT mutation was absent in 33 of 59 cases. However, 9 cases in the flow cytometry negative group fulfilled the criteria, other than aberrant CD2 and/or CD25 expression, for the diagnosis of systemic mastocytosis. Assessment of Flow Cytometric Data in All Cases of Systemic Mastocytosis Table 4 summarizes flow cytometric results for all cases with the clinical diagnosis of systemic mastocytosis (n = 39) 420 Am J Clin Pathol 2012;138: DOI: /AJCP5PJWK4QFHWHM

6 Hematopathology / Original Article based on the pathologic and/or laboratory findings according to the 8 WHO criteria. Initial flow cytometric analysis of these cases revealed 18 positive, 11 suspicious, and 10 negative results, based on aberrant CD2 and/or CD25 expression. Detailed analysis of the flow cytometric data from these cases showed that 24 cases (62%) had a discrete and expanded population of mast cells within the CD117-bright mast cell gate Image 3 even in the absence of an aberrant immunophenotype. In the majority of these cases (n = 17; 71%), mast cells had an aberrant immunophenotype, demonstrating either CD25 expression or CD2/CD25 expression. However, 7 cases (29%) did not demonstrate aberrant expression of CD2 and/or CD25 and originally were interpreted as negative for the presence of a mast cell neoplasm. Further investigation of these cases revealed that all of them had a bone marrow biopsy specimen that was morphologically positive for systemic mastocytosis, a bone marrow aspirate smear that was positive for mast cell disease, and an elevated serum tryptase level. KIT mutation analysis was not performed. All 7 cases had a clinical diagnosis of systemic mastocytosis. Based on the % correlation between the presence of a discrete CD117-bright population of mast cells on flow cytometric analysis (even in the absence of an aberrant immunophenotype) and positive biopsy morphologic findings and laboratory findings, we revised our flow cytometric criteria for the presence of mast cell disease to include a discrete CD117- bright population as a positive criterion. A 2 1 Correlation of Flow Cytometric Results Based on the Revised Criteria With Bone Marrow Biopsy, Laboratory, and Clinical Data Based on our data, we revised the flow cytometric criteria for evaluation of a mast cell neoplasm. The results were then interpreted as positive based on the presence of a discrete population of CD117-bright cells and/or aberrant CD2 and/ or CD25 expression. Criteria for suspicious and negative flow cytometric findings remained unchanged. When the revised flow cytometric criteria were applied to the previously analyzed bone marrow samples, 3 suspicious and 7 negative cases were reclassified as positive, increasing the positive flow cytometry group by 10 cases (Table 2). These revised flow cytometric results were again correlated with morphologic and laboratory data Table 5. Then the flow cytometry positive/suspicious group constituted 41 cases and the flow cytometry negative group contained 52 cases. Based on the new flow cytometric criteria, the sensitivity of the test was increased, and only 2 cases of systemic mastocytosis were missed on flow cytometric analysis. All other flow cytometric cases had a good correlation between the biopsy and other laboratory and clinical findings. Comparison of Sensitivity and Specificity of 2 Flow Cytometric Analyses Sensitivity and specificity were calculated using the standard and revised flow cytometric criteria. The standard B 2 1 Image 3 Examples of positive flow cytometric analyses based on either aberrant immunophenotype (A) or presence of an expanded discrete population within the CD117-bright gate (B). Am J Clin Pathol 2012;138: DOI: /AJCP5PJWK4QFHWHM 421

7 Pozdnyakova et al / New Mastocytosis Flow Cytometry Approach Table 5 Correlation of Revised Flow Cytometry Results With Other Concurrent Results* Other Results Elevated Tryptase KIT D816V Systemic Flow Cytometry Results Biopsy Levels (>20 ng/ml) Mutation Mastocytosis Positive/suspicious for mast cell neoplasm (n = 41) Positive, n Negative, n Not available Negative for mast cell neoplasm (n = 52) Positive, n Negative, n Not available, n * Other concurrent results include bone marrow biopsy findings, tryptase levels, KIT D816V mutation status, and clinical diagnosis of systemic mastocytosis. Data are given as numbers of cases. One patient with the diagnosis of mast cell sarcoma carried codon 419 mutation. flow cytometric criteria included only aberrant CD2 and/or CD25 expression by the mast cells. The revised criteria included aberrant CD2 and/or CD25 expression by the mast cells and/or presence of a discrete and expanded population of mast cells based on CD117-bright expression. The results are summarized in Table 6. Sensitivity of flow cytometric analysis increased from 77% to 95%, and exceeded the sensitivity of morphologic analyses of the bone marrow aspirate smear (69%) or the bone marrow biopsy specimen (85%). The revised flow cytometric criteria did not alter the specificity of the test, which was 96%, and was similar to that for the bone marrow biopsy and aspirate (98%). Discussion One of the minor 8 WHO criteria for the diagnosis of systemic mastocytosis is aberrant expression of CD2 and/ or CD25 by neoplastic mast cells. 1 The immunophenotypic evaluation for aberrant expression can be performed with either immunohistochemical or flow cytometric analysis with both methods reporting similar sensitivity. 9 However, flow cytometry uses a multiparametric approach that allows the analysis of aberrant marker(s) expression on specifically gated mast cells. This approach is essential in cases in which the biopsy shows no discrete and/or large aggregates of mast cells. Thus immunohistochemical assessment for CD2 and/ or CD25 is difficult, because the expression of these markers is not unique to mast cells, but is seen on lymphocytes and other activated cells. 5 In the past several decades, flow cytometric immunophenotyping of mast cells has become a useful adjunctive test in the evaluation for systemic mastocytosis, with current criteria of positivity based on aberrant expression of CD2 and/or CD25 by the mast cells. 1,5,7 Absence of a standard flow cytometric approach for the evaluation of mastocytosis cases has led to controversy regarding the frequency Table 6 Comparison of Sensitivity and Specificity Between Flow Cytometry, Aspirate, and Biopsy Findings in Systemic Mastocytosis Flow Cytometry Standard Revised Biopsy Aspirate Sensitivity, % Specificity, % of aberrant marker(s) expression on the neoplastic mast cells and the usefulness of CD2 as an aberrant marker. 8 One of the limitations of the flow cytometric immunophenotypic assessment for systemic mastocytosis is the low frequency of mast cells in bone marrow samples. Our approach to address this problem was to collect at least 1,000,000 events for analysis from bone marrow aspirate samples whenever possible. We then prospectively analyzed 93 consecutive bone marrow samples that were sent to our laboratory for evaluation for systemic mastocytosis. When the sample was limited and 1,000,000 events could not be collected, the entire specimen was analyzed. The mean number of collected events was 580,000 with most samples having more than 400,000 events for analysis (data not shown). Acquisition of a large number of events allowed us to observe a discrete population of mast cells within the mast cell gate that was based on side scatter and CD117-bright expression (Image 3). Cases that exhibited a discrete population of mast cells had a higher mean number of mast cells than cases without a discrete mast cell population (10,336 vs 296, respectively, data not shown). A discrete population of mast cells was observed in 24 cases and included cases with aberrant CD2 and/or CD25 expression as well as cases without aberrant expression of these markers (Table 4). All cases that exhibited a discrete population of mast cells on flow cytometric analysis had a 422 Am J Clin Pathol 2012;138: DOI: /AJCP5PJWK4QFHWHM

8 Hematopathology / Original Article clinical diagnosis of systemic mastocytosis based on other diagnostic criteria. We conclude that the presence of a discrete population of CD117-positive mast cells on flow cytometric analysis is an indicator of the presence of a mast cell neoplasm and may be a useful new diagnostic criterion for systemic mastocytosis, even in the absence of an aberrant mast cell immunophenotype. Additional studies at other institutions are needed to confirm our observation. Using revised flow cytometric criteria for involvement by a mast cell neoplasm that included the presence of a discrete mast cell population and/ or aberrant immunophenotype, the sensitivity of flow cytometric analysis for the detection of mast cell disease increased from 77% to 95%, with no decrease in the specificity of the analysis (Table 6). This difference in sensitivity between the 2 methods was a result of 7 cases with flow cytometric findings that were interpreted as positive and biopsy findings that were interpreted as negative because of a lack of mast cell aggregates. In the absence of mast cell aggregates, morphologic assessment of mast cells for the presence of atypical forms and immunohistochemical analysis for aberrant marker(s) expression become less reliable and more subjective, and in these cases, flow cytometric analysis provides more sensitive and objective results. Based on the revised flow cytometric criteria, 41 cases were interpreted as positive or suspicious for involvement by a mast cell disorder. Thirty-nine of these cases had a clinical diagnosis of systemic mastocytosis, 1 case had a diagnosis of cutaneous mastocytosis, and 1 case had a diagnosis of recurrent anaphylaxis. The majority of these cases (n = 34, 83%) showed an aberrant immunophenotype, with CD2/CD25 coexpression in 24 cases (70%), CD25 expression in 9 cases (26%), and CD2 expression in 1 case (3%). Seven (17%) of 41 cases did not show an aberrant immunophenotype. Our results support the findings that CD25 is a more informative diagnostic marker than CD2 and its expression alone or in combination with CD2 is seen in more than 80% of cases. 8 In addition, our series showed that the aberrant immunophenotype in mast cell neoplasms is seen in approximately 85% of cases. Another observation we made was the persistence of the immunophenotype of the neoplastic mast cell population. We studied 5 patients with systemic mastocytosis with sequential follow-up which included flow cytometric analysis. In all 5 patients, the immunophenotype of the neoplastic mast cells did not change over time. Three patients had aberrant expression of both CD2 and CD25, 1 patient demonstrated expression of only CD25, and 1 patient showed a discrete mast cell population without aberrant expression of either CD2 or CD25. Because flow cytometry is an exceptionally sensitive method for detecting neoplastic mast cells, even with a low burden of the disease, one would expect to identify cases that do not fulfill all diagnostic criteria for systemic mastocytosis, ie, monoclonal mast cell activation syndrome. A diagnosis of monoclonal mast cell activation syndrome has been accepted for patients with anaphylaxis or other mast cell activation symptoms that meet 1 of 2 minor diagnostic criteria for systemic mastocytosis but do not demonstrate mast cell aggregates in the extracutaneous organs. 11,12 In our series, we evaluated 8 patients with the clinical diagnosis of monoclonal mast cell activation syndrome. Although bone marrow biopsy in these patients did not demonstrate mast cell aggregates, flow cytometry demonstrated aberrant CD25 and/ or CD2 expression in 2 patients. One patient had a clinical diagnosis of cutaneous mastocytosis, was positive for the KIT D816V mutation, and showed a small population (249 events) of CD25-positive mast cells on flow cytometric analysis. A second patient presented with a history of recurrent anaphylactic reactions, and on flow cytometry, was found to have a small mast cell clone (413 events) with aberrant coexpression of CD2 and CD25; KIT D816V analysis was negative. Interestingly, neither of these patients showed a discrete CD117-bright mast cell population on flow cytometry, confirming our observation that the presence of a discrete mast cell population on flow cytometric analysis correlates with the presence of mast cell aggregates on a biopsy and the diagnosis of systemic mastocytosis. Other cases of monoclonal mast cell activation syndrome had negative flow cytometric results that can be explained by the low number of events collected. Of 8 patients with a clinical diagnosis of monoclonal mast cell activation syndrome, 1,000,000 events were collected in only 3. These cases included 2 with an aberrant mast cell immunophenotype. The number of events collected for the remaining 5 cases ranged from,000 to 5,000. We believe that increased number of collected events will lead to recognition of more cases of monoclonal mast cell activation syndrome on flow cytometry. Similarly, we had 2 cases with the clinical diagnosis of indolent systemic mastocytosis that were negative on flow cytometric analysis; neither a discrete mast cell population nor aberrant immunophenotype was seen in these cases. Biopsy showed both cases having a low disease burden, with 1 specimen interpreted as negative and 1 as suspicious based on aberrant CD25 expression and 2 small mast cell clusters of fewer than 15 cells. The samples for flow cytometric analysis were limited in both cases and fewer than 1,000,000 events were collected (,000 and 525,000 events). The diagnosis of systemic mastocytosis was made based on the minor criteria of elevated tryptase levels and positivity for KIT D816V mutation. In conclusion, we developed an optimized flow cytometric method for evaluating patients with mast cell neoplasms. Large number of collected events for analysis allowed a discrete mast cell population to be identified in patients with Am J Clin Pathol 2012;138: DOI: /AJCP5PJWK4QFHWHM 423

9 Pozdnyakova et al / New Mastocytosis Flow Cytometry Approach systemic mastocytosis, even in the absence of an aberrant immunophenotype, and predicted bone marrow involvement by the neoplastic mast cells on the biopsy. In addition, in patients with recurrent episodes consistent with mast cell activation that do not fulfill all criteria for systemic mastocytosis, high-sensitivity flow cytometric analysis allowed detection of small clonal populations of mast cells leading to a diagnosis of monoclonal mast cell activation syndrome. The sensitivity of the revised flow cytometric analysis exceeds the sensitivity of the bone marrow biopsy, currently considered the gold standard, thus making flow cytometric analysis an excellent diagnostic test for mast cell neoplasms. From the 1 Department of Pathology, Brigham and Women s Hospital, Harvard Medical School, Boston, MA; and 2 Department of Pathology, University Pathologists at Southcoast Hospitals Group, St. Luke s Hospital, New Bedford, MA. Address reprint requests to Dr Pozdnyakova: Department of Pathology, Brigham and Women s Hospital, Harvard Medical School, 75 Francis St, Amory 3, Boston, MA 02115; opozdnyakova@partners.org. References 1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, France: IARC Press; Valent P, Horny HP, Escribano L, et al. Diagnostic criteria and classification of mastocytosis: a consensus proposal. Leuk Res. 1;25: Lennert K, Parwaresch MR. Mast cells and mast cell neoplasia: a review. Histopathology. 1979;3: Longley BJ, Reguera MJ, Ma Y. Classes of c-kit activating mutations: proposed mechanisms of action and implications for disease classification and therapy. Leuk Res. 1;25: Escribano L, Diaz-Agustin B, Bellas C, et al. Utility of flow cytometric analysis of mast cells in the diagnosis and classification of adult mastocytosis. Leuk Res. 1;25: Escribano L, Orfao A, Díaz-Agustin B, et al. Indolent systemic mast cell disease in adults: immunophenotypic characterization of bone marrow mast cells and its diagnostic implications. Blood. 1998;91: Escribano L, Diaz Agustin B, Bravo P, et al. Immunophenotype of bone marrow mast cells in indolent systemic mast cell disease in adults. Leuk Lymphoma. 1999;35: Morgado JMT, Sanchez-Munoz L, Teodosio CG, et al. Immunophenotyping in systemic mastocytosis diagnosis: CD25 positive alone is more informative than the CD25 and/or CD2 WHO criterion [published online ahead of print January 6, 2012]. 2012;25: Baumgartner C, Sonneck K, Krauth MT, et al. Immunohistochemical assessment of CD25 is equally sensitive and diagnostic in mastocytosis compared to flow cytometry. Eur J Clin Invest. 8;38: Lowry R. Clinical calculator 1. Available at: vassar.edu/lowry/clin1.html. Accessed June 28, Valent P, Akin C, Escribano L, et al. Standards and standardization in mastocytosis: Consensus Statements on Diagnostics, Treatment Recommendations and Response Criteria. Eur J Clin Invest. 7;37: Akin C, Scott LM, Kocabas CN, et al. Demonstration of an aberrant mast-cell population with clonal markers in a subset of patients with idiopathic anaphylaxis. Blood. 7;110: Am J Clin Pathol 2012;138: DOI: /AJCP5PJWK4QFHWHM