Letter to the Editor

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Blood Res 2018; 53(3):

Published online September 28, 2018

https://doi.org/10.5045/br.2018.53.3.251

© The Korean Society of Hematology

Massive splenomegaly: flow cytometry as a diagnostic tool for systemic mastocytosis

Loveena Rastogi1, Jasmita Dass1*, Gaurav Dhingra1, Nitin Gupta2, and Jyoti Kotwal1

1Department of Hematology, Sir Ganga Ram Hospital, New Delhi, India.

2Department of Clinical Hematology, Sir Ganga Ram Hospital, New Delhi, India.

Correspondence to : Jasmita Dass Department of Hematology, Sir Ganga Ram Hospital, New Delhi 110060, India. drjasmita@gmail.com

Received: February 2, 2018; Revised: March 27, 2018; Accepted: May 10, 2018

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

TO THE EDITOR: Mastocytosis is a spectrum of disorders related to the proliferation and accumulation of mast cells in one or more organs. Systemic mastocytosis (SM) was classified by World Health Organisation (WHO) classification for hematolymphoid neoplasms in 2008 as a myeloproliferative neoplasm. However, in the 2016 update of the WHO classification, it has been regarded as a separate entity. SM is sub-classified into indolent SM, smoldering SM, SM with an associated hematological neoplasm (AHN), aggressive SM, and mast cell leukemia [1]. SM is characterized by involvement of at least one extracutaneous organ with or without evidence of skin lesions [2]. We are presenting a rare case of aggressive SM with massive splenomegaly, which could be diagnosed on bone marrow (BM) immunophenotyping.

A 61-year-old man presented with a 2-year history of progressively increasing left hypochondriac lump, abdominal pain, and progressive weight loss of 25–30 kg in the last 3 months. There was no fever, night sweats, blood in stools, hematemesis, bony pain, or bleeding from any other site. He was an occasional alcoholic and non-smoker. On physical examination, the patient was afebrile with normal vital signs. No pallor, icterus, clubbing, cyanosis, edema, and peripheral lymphadenopathy were found. Abdominal examination revealed massive splenomegaly (16 cm below the left coastal margin) and hepatomegaly (4 cm below the right coastal margin), with the rest of the systemic examination within normal limits. The complete blood count showed hemoglobin level of 10.2 g/dL, total leukocyte count of 5,480/µL, and platelet count of 282,000/µL. Differential count revealed neutrophils 70%, lymphocytes 11%, monocytes 4%, metamyelocyte 1%, basophil 1%, and eosinophils 13%. The absolute eosinophil count was 712/µL, and lactate dehydrogenase (LDH) level was 180 U/L (laboratory reference range is 91–180 U/L). HIV/HBsAg/HCV were negative. Liver and renal function tests were normal. Stool examination was unremarkable. Abdominal ultrasonography showed massive splenomegaly with hepatomegaly and normal echotexture of the liver.

In view of marked hepatosplenomegaly, BM aspiration and biopsy were done. BM aspiration showed hypercellular marrow with myeloid predominance and prominent eosinophils and their precursors constituting 23% of the myelogram. Blasts were not increased. Mast cells were increased in the aspirate and imprint smears and highlighted by toluidine blue staining (Fig. 1A). Some mast cells exhibited hypogranularity and oval nuclei, instead of normal round (Fig. 1A inset). The overall mast cell percentage was −8%. The flow cytometric (FCM) analysis of the BM aspirate showed aberrant immunophenotype in mast cells. The CD117-positive mast cells showed aberrant expression of CD2 and CD25. In addition, they exhibited slight downregulation of CD117 (Fig. 1B–E). BM biopsy showed 80–90% cellularity with marked eosinophilic hyperplasia and increased megakaryocytes. There were multiple large aggregates of mast cells found mostly around the perivascular and paratrabecular regions. The paratrabecular aggregates were causing new bone formation (Fig. 2A). Each aggregate comprised more than 15 cells, with >25% exhibiting atypical morphology (Fig. 2B). The reticulin staining showed grade 2 fibrosis. Toluidine blue staining was performed on BM aspiration and biopsy, which highlighted the mast cell hyperplasia (Fig. 2C). Immunohistochemistry (IHC) for CD117 was performed, and mast cells were found to be positive (Fig. 2D). The reverse transcriptase- polymerase chain reaction (RT-PCR) for FIP1L1-PDGFRA was negative. However, KITD816V mutation and serum tryptase could not be performed as the patient was hesitant. Further, a history of diarrhea was elicited from the patient.

Therefore, the patient fulfilled the major criteria of the presence of dense mast cell aggregates and the minor criteria of atypical morphology and abnormal immunophenotype and was, hence, classified as having SM. Further, he had three “B” findings in the form of presence of splenomegaly without hypersplenism, hepatomegaly without ascites, and myeloproliferation. Moreover, the history of diarrhea and weight loss pointed toward the presence of one C finding of malabsorption.

Considering all the above findings and investigations, we diagnosed our patient with aggressive SM. The patient was initially administered with interferon alpha but was lost to follow-up.

Mastocytosis is an infrequent condition characterized by abnormal growth and accumulation of mast cells in various organs. This condition is subdivided into cutaneous mastocytosis (CM) and SM [2]. CM is more frequent during childhood and usually presents at an early age usually in the first year of life. SM involves at least one extracutaneous organ and may follow an indolent course or may be associated with life-threatening symptoms. The BM is the most frequently involved organ in SM [3]. The diagnosis of SM requires one major and one minor criteria of classification of WHO 2008. Further sub-classification of SM requires the presence of “B” and “C” findings. One of the minor WHO 2008 criteria for the diagnosis of SM is aberrant expression of CD2 and/or CD25 by neoplastic mast cells [2]. These diagnostic criteria have remained the same in the WHO 2016 classification [1].

In the past few years, immunophenotyping of mast cells has become a useful adjunct in the evaluation of SM. The normal mast cells are positive for CD117 and consistently negative for CD2 and CD25. In SM, the mast cells are commonly positive for CD2, CD25, or both. Although both IHC and FCM analysis can be performed, IHC assessment for CD2 and/or CD25 is difficult, because lymphocytes and few other activated cells also express these markers. However, FCM uses a multiparametric approach that allows the aberrant expression of the above markers on specifically gated mast cells [4], and this was applied in our case. Thus, these markers distinguish neoplastic mast cells from normal and reactive mast cells as they both do not express CD2 and CD25 [5].

Being an extremely sensitive method, FCM is useful in detecting neoplastic mast cells even with a low burden of the disease and expected to identify even those cases which do not fulfill the diagnostic criteria for SM [4]. It proved to be an important diagnostic tool in the present case as mast cells may be increased in many reactive conditions as well.

The diagnosis of SM requires a cognisance of this rare entity along with a high index of suspicion. It should be considered as one of the differential diagnoses in cases with massive splenomegaly especially when the usual causes have been excluded. The assessment of the immunophenotype of mast cells is helpful in establishing the diagnosis of SM. This case also highlights the role of FCM in identifying abnormal mast cells based on routinely available markers in laboratories performing leukemia FCM.

Fig. 1.

(A) Toluidine blue staining shows mast cells in a marrow particle (toluidine blue, ×200). The inset shows spindle cell morphology of mast cells (H&E, ×400). (B) CD45 vs side scatter plot (SSC) to gate lymphocytes (reddish brown). (C) Gating for mast cells was done on CD117 vs SSC dot plot. Bright CD117+ cells are gated as mast cells (dark blue). (D, E) Dot plots and color density plots respectively showing an aberrant expression of CD2 and CD25 on the mast cell population. The intensity of CD2 is lesser than the normal T lymphocytes.


Fig. 2.

(A) Paratrabecular mast cell aggregate showing new bone formation (H&E, ×100). (B) A high-power view shows the atypical morphology of mast cells with a predominance of oval- and spindle-shaped mast cells. Eosinophils are interspersed in the aggregates (H&E, ×400). (C) Toluidine blue staining shows metachromasia in mast cell aggregates (×200). (D) CD117 immunohistochemistry on bone marrow biopsy is positive in paratrabecular mast cell aggregates (×200).


  1. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016;127:2391-2405.
    Pubmed
  2. Horny HP, Metcalfe DD, Bennett JM, et al. In: Swerdlow SH, Campo E, Harris NL. WHO classification of tumours of haematopoietic and lymphoid tissues. 4th ed. Lyon, France: IARC Press; 2008. p. 54-63.
  3. Arredondo AR, Gotlib J, Shier L, et al. Myelomastocytic leukemia versus mast cell leukemia versus systemic mastocytosis associated with acute myeloid leukemia: a diagnostic challenge. Am J Hematol 2010;85:600-606.
    Pubmed
  4. Pozdnyakova O, Kondtratiev S, Li B, Charest K, Dorfman DM. High-sensitivity flow cytometric analysis for the evaluation of systemic mastocytosis including the identification of a new flow cytometric criterion for bone marrow involvement. Am J Clin Pathol 2012;138:416-424.
    Pubmed
  5. Jabbar KJ, Medeiros LJ, Wang SA, et al. Flow cytometric immunophenotypic analysis of systemic mastocytosis involving bone marrow. Arch Pathol Lab Med 2014;138:1210-1214.
    Pubmed

Article

Letter to the Editor

Blood Res 2018; 53(3): 251-254

Published online September 28, 2018 https://doi.org/10.5045/br.2018.53.3.251

Copyright © The Korean Society of Hematology.

Massive splenomegaly: flow cytometry as a diagnostic tool for systemic mastocytosis

Loveena Rastogi1, Jasmita Dass1*, Gaurav Dhingra1, Nitin Gupta2, and Jyoti Kotwal1

1Department of Hematology, Sir Ganga Ram Hospital, New Delhi, India.

2Department of Clinical Hematology, Sir Ganga Ram Hospital, New Delhi, India.

Correspondence to: Jasmita Dass Department of Hematology, Sir Ganga Ram Hospital, New Delhi 110060, India. drjasmita@gmail.com

Received: February 2, 2018; Revised: March 27, 2018; Accepted: May 10, 2018

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Case Report

TO THE EDITOR: Mastocytosis is a spectrum of disorders related to the proliferation and accumulation of mast cells in one or more organs. Systemic mastocytosis (SM) was classified by World Health Organisation (WHO) classification for hematolymphoid neoplasms in 2008 as a myeloproliferative neoplasm. However, in the 2016 update of the WHO classification, it has been regarded as a separate entity. SM is sub-classified into indolent SM, smoldering SM, SM with an associated hematological neoplasm (AHN), aggressive SM, and mast cell leukemia [1]. SM is characterized by involvement of at least one extracutaneous organ with or without evidence of skin lesions [2]. We are presenting a rare case of aggressive SM with massive splenomegaly, which could be diagnosed on bone marrow (BM) immunophenotyping.

A 61-year-old man presented with a 2-year history of progressively increasing left hypochondriac lump, abdominal pain, and progressive weight loss of 25–30 kg in the last 3 months. There was no fever, night sweats, blood in stools, hematemesis, bony pain, or bleeding from any other site. He was an occasional alcoholic and non-smoker. On physical examination, the patient was afebrile with normal vital signs. No pallor, icterus, clubbing, cyanosis, edema, and peripheral lymphadenopathy were found. Abdominal examination revealed massive splenomegaly (16 cm below the left coastal margin) and hepatomegaly (4 cm below the right coastal margin), with the rest of the systemic examination within normal limits. The complete blood count showed hemoglobin level of 10.2 g/dL, total leukocyte count of 5,480/µL, and platelet count of 282,000/µL. Differential count revealed neutrophils 70%, lymphocytes 11%, monocytes 4%, metamyelocyte 1%, basophil 1%, and eosinophils 13%. The absolute eosinophil count was 712/µL, and lactate dehydrogenase (LDH) level was 180 U/L (laboratory reference range is 91–180 U/L). HIV/HBsAg/HCV were negative. Liver and renal function tests were normal. Stool examination was unremarkable. Abdominal ultrasonography showed massive splenomegaly with hepatomegaly and normal echotexture of the liver.

In view of marked hepatosplenomegaly, BM aspiration and biopsy were done. BM aspiration showed hypercellular marrow with myeloid predominance and prominent eosinophils and their precursors constituting 23% of the myelogram. Blasts were not increased. Mast cells were increased in the aspirate and imprint smears and highlighted by toluidine blue staining (Fig. 1A). Some mast cells exhibited hypogranularity and oval nuclei, instead of normal round (Fig. 1A inset). The overall mast cell percentage was −8%. The flow cytometric (FCM) analysis of the BM aspirate showed aberrant immunophenotype in mast cells. The CD117-positive mast cells showed aberrant expression of CD2 and CD25. In addition, they exhibited slight downregulation of CD117 (Fig. 1B–E). BM biopsy showed 80–90% cellularity with marked eosinophilic hyperplasia and increased megakaryocytes. There were multiple large aggregates of mast cells found mostly around the perivascular and paratrabecular regions. The paratrabecular aggregates were causing new bone formation (Fig. 2A). Each aggregate comprised more than 15 cells, with >25% exhibiting atypical morphology (Fig. 2B). The reticulin staining showed grade 2 fibrosis. Toluidine blue staining was performed on BM aspiration and biopsy, which highlighted the mast cell hyperplasia (Fig. 2C). Immunohistochemistry (IHC) for CD117 was performed, and mast cells were found to be positive (Fig. 2D). The reverse transcriptase- polymerase chain reaction (RT-PCR) for FIP1L1-PDGFRA was negative. However, KITD816V mutation and serum tryptase could not be performed as the patient was hesitant. Further, a history of diarrhea was elicited from the patient.

Therefore, the patient fulfilled the major criteria of the presence of dense mast cell aggregates and the minor criteria of atypical morphology and abnormal immunophenotype and was, hence, classified as having SM. Further, he had three “B” findings in the form of presence of splenomegaly without hypersplenism, hepatomegaly without ascites, and myeloproliferation. Moreover, the history of diarrhea and weight loss pointed toward the presence of one C finding of malabsorption.

Considering all the above findings and investigations, we diagnosed our patient with aggressive SM. The patient was initially administered with interferon alpha but was lost to follow-up.

Discussion

Mastocytosis is an infrequent condition characterized by abnormal growth and accumulation of mast cells in various organs. This condition is subdivided into cutaneous mastocytosis (CM) and SM [2]. CM is more frequent during childhood and usually presents at an early age usually in the first year of life. SM involves at least one extracutaneous organ and may follow an indolent course or may be associated with life-threatening symptoms. The BM is the most frequently involved organ in SM [3]. The diagnosis of SM requires one major and one minor criteria of classification of WHO 2008. Further sub-classification of SM requires the presence of “B” and “C” findings. One of the minor WHO 2008 criteria for the diagnosis of SM is aberrant expression of CD2 and/or CD25 by neoplastic mast cells [2]. These diagnostic criteria have remained the same in the WHO 2016 classification [1].

In the past few years, immunophenotyping of mast cells has become a useful adjunct in the evaluation of SM. The normal mast cells are positive for CD117 and consistently negative for CD2 and CD25. In SM, the mast cells are commonly positive for CD2, CD25, or both. Although both IHC and FCM analysis can be performed, IHC assessment for CD2 and/or CD25 is difficult, because lymphocytes and few other activated cells also express these markers. However, FCM uses a multiparametric approach that allows the aberrant expression of the above markers on specifically gated mast cells [4], and this was applied in our case. Thus, these markers distinguish neoplastic mast cells from normal and reactive mast cells as they both do not express CD2 and CD25 [5].

Being an extremely sensitive method, FCM is useful in detecting neoplastic mast cells even with a low burden of the disease and expected to identify even those cases which do not fulfill the diagnostic criteria for SM [4]. It proved to be an important diagnostic tool in the present case as mast cells may be increased in many reactive conditions as well.

Conclusion

The diagnosis of SM requires a cognisance of this rare entity along with a high index of suspicion. It should be considered as one of the differential diagnoses in cases with massive splenomegaly especially when the usual causes have been excluded. The assessment of the immunophenotype of mast cells is helpful in establishing the diagnosis of SM. This case also highlights the role of FCM in identifying abnormal mast cells based on routinely available markers in laboratories performing leukemia FCM.

Fig 1.

Figure 1.

(A) Toluidine blue staining shows mast cells in a marrow particle (toluidine blue, ×200). The inset shows spindle cell morphology of mast cells (H&E, ×400). (B) CD45 vs side scatter plot (SSC) to gate lymphocytes (reddish brown). (C) Gating for mast cells was done on CD117 vs SSC dot plot. Bright CD117+ cells are gated as mast cells (dark blue). (D, E) Dot plots and color density plots respectively showing an aberrant expression of CD2 and CD25 on the mast cell population. The intensity of CD2 is lesser than the normal T lymphocytes.

Blood Research 2018; 53: 251-254https://doi.org/10.5045/br.2018.53.3.251

Fig 2.

Figure 2.

(A) Paratrabecular mast cell aggregate showing new bone formation (H&E, ×100). (B) A high-power view shows the atypical morphology of mast cells with a predominance of oval- and spindle-shaped mast cells. Eosinophils are interspersed in the aggregates (H&E, ×400). (C) Toluidine blue staining shows metachromasia in mast cell aggregates (×200). (D) CD117 immunohistochemistry on bone marrow biopsy is positive in paratrabecular mast cell aggregates (×200).

Blood Research 2018; 53: 251-254https://doi.org/10.5045/br.2018.53.3.251

References

  1. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016;127:2391-2405.
    Pubmed
  2. Horny HP, Metcalfe DD, Bennett JM, et al. In: Swerdlow SH, Campo E, Harris NL. WHO classification of tumours of haematopoietic and lymphoid tissues. 4th ed. Lyon, France: IARC Press; 2008. p. 54-63.
  3. Arredondo AR, Gotlib J, Shier L, et al. Myelomastocytic leukemia versus mast cell leukemia versus systemic mastocytosis associated with acute myeloid leukemia: a diagnostic challenge. Am J Hematol 2010;85:600-606.
    Pubmed
  4. Pozdnyakova O, Kondtratiev S, Li B, Charest K, Dorfman DM. High-sensitivity flow cytometric analysis for the evaluation of systemic mastocytosis including the identification of a new flow cytometric criterion for bone marrow involvement. Am J Clin Pathol 2012;138:416-424.
    Pubmed
  5. Jabbar KJ, Medeiros LJ, Wang SA, et al. Flow cytometric immunophenotypic analysis of systemic mastocytosis involving bone marrow. Arch Pathol Lab Med 2014;138:1210-1214.
    Pubmed
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