Year : 2009 | Volume
: 52 | Issue : 4 | Page : 490--494
Megakaryocytic alterations in thrombocytopenia: A bone marrow aspiration study
Manas Muhury, Alka M Mathai, Sharada Rai, Ramadas Naik, Muktha R Pai, Ruchi Sinha
Department of Pathology, Kasturba Medical College, Mangalore, Karnataka, India
Department of Pathology, Kasturba Medical College, Mangalore-575 001, Karnataka
Context: Dysplastic changes are well documented in myelodysplastic syndromes (MDS). However, they are also observed in non-MDS hematological conditions. Aims: To evaluate the megakaryocytic alterations in the bone marrow aspirations in cases of non-MDS related thrombocytopenia. Setting and Design: A prospective study of 144 bone marrow aspirates was conducted in the department of pathology, Kasturba Medical College, Mangalore. The aspirates were studied to assess the number and morphology of the megakaryocytes in non-MDS related thrombocytopenia and evaluate their significance when compared to changes in MDS. Materials and Methods: The bone marrow aspiration smears were stained with Leishman stain and examined under light microscope. Statistical Analysis Used: Fisher«SQ»s exact test. A P value less than 0.05 was considered significant. Sensitivity and specificity was calculated for those features which were significant in the relevant hematological disorders. Results: The sensitivity of immature megakaryocytes, dysplastic forms and micromegakaryocytes in cases of immune thrombocytopenic purpura was 100%, 89% and 42% respectively. The specificity of emperipolesis was 74%. In cases of infection-associated thrombocytopenia, immature megakaryocytes had a sensitivity of 100% and cytoplasmic vacuolization were 86% specific. The sensitivity of the dysplastic forms in megaloblastic anemia was 75%. However, no platelet budding was observed. The presence of micromegakaryocyte had a specificity of 83% in MDS, and was statistically significant when compared to cases of non-MDS conditions (P<0.05). Conclusions: Careful understanding of the morphological changes of megakaryocytes in bone marrow aspirates can improve the diagnostic accuracy for a wide range of hematological disorders thereby enabling proper therapeutic interventions.
|How to cite this article:|
Muhury M, Mathai AM, Rai S, Naik R, Pai MR, Sinha R. Megakaryocytic alterations in thrombocytopenia: A bone marrow aspiration study.Indian J Pathol Microbiol 2009;52:490-494
|How to cite this URL:|
Muhury M, Mathai AM, Rai S, Naik R, Pai MR, Sinha R. Megakaryocytic alterations in thrombocytopenia: A bone marrow aspiration study. Indian J Pathol Microbiol [serial online] 2009 [cited 2020 Feb 20 ];52:490-494
Available from: http://www.ijpmonline.org/text.asp?2009/52/4/490/56132
Thrombocytopenia is commonly encountered in various hematological disorders including myelodysplastic syndromes (MDS) as well as various non-myelodysplastic hematological conditions.  Dysplastic changes are well known in megakaryocytes in thrombocytopenia associated with MDS. However, they are also observed in megakaryocytes in non-myelodysplastic hematological conditions also; but scant data exist on the prevalence of dysplastic changes in megakaryocytes in non-myelodysplastic hematological conditions. This study was undertaken for better understanding of the dysplastic megakaryocytic alterations and their contribution to thrombocytopenia in non-MDS diseases so as to increase the diagnostic accuracy.
Materials and Methods
A prospective study of 144 bone marrow aspirates was conducted. The non-MDS hematological disorders with thrombocytopenia included in the study were cases of immune thrombocytopenic purpura (ITP), infection-associated thrombocytopenia (IAT), hypersplenism, aplastic anemia, dimorphic anemia, megaloblastic anemia, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), lymphoma-leukemia syndrome (LLS), myeloma, bone marrow metastasis and blast crisis of chronic myeloid leukemia (CML). The clinical details, complete blood counts, and other relevant laboratory investigations were obtained.
The bone marrow smears were stained with Leishman stain and examined for changes in the number and morphology of megakaryocytes in thrombocytopenia which was defined as platelet count less than 1,50,000/mm 3 . The number of the megakaryocytes was considered as normal (one megakaryocyte per one to three low-power fields), increased (more than two megakaryocytes per low-power field) or decreased (one megakaryocyte per five to ten low-power fields).  The morphological changes of megakaryocytes that were studied included nuclear segmentation, presence of immature forms, dysplastic forms, micromegakaryocytes, emperipolesis, platelet budding, cytoplasmic vacuolization, bare megakaryocytic nuclei and hypogranular forms. The presence of abnormal megakaryocytes which included the micromegakaryocytes, dysplastic forms, megakaryocytes with separated lobes and hypogranular forms were considered as dysmegakaryocytopoiesis. Normal megakaryocytes were considered to have four to sixteen nuclear lobes. Immature megakaryocytes were defined as young forms of megakaryocytes with scant bluish cytoplasm and lacking lobulation of the nucleus which occupied most of the cell.  Dysplastic megakaryocytes were defined as those with single/ multiple separate nuclei. Micromegakaryocytes were defined as megakaryocytes whose size was that of a large lymphocyte/monocyte and which had a single/bilobed nucleus. The megakaryocytes were considered to show platelet budding if there was budding of cytoplasmic processes from their surfaces. Hypogranular forms were defined as megakaryocytes with pale grey or water clear cytoplasm and sparse or no granules. The type of cell seen within the megakaryocyte in emperipolesis was also documented.
The number and morphology of the megakaryocytes in non-MDS related thrombocytopenia were assessed. Their significance was studied by comparing with the morphological changes in MDS. The distribution of morphological changes in cases of non-MDS conditions and MDS were compared using Fisher's exact test. A P value less than 0.05 was considered significant. The sensitivity and specificity for those morphological features which were significant in the relevant hematological disorders was also calculated.
The commonest cause of thrombocytopenia for which bone marrow examination was sought was AML (27 cases, 18.8%). The second most common cause was ITP (19 cases, 13.15%) which was followed by ALL and dimorphic anemia (18 cases each, 12.5%). There were 17 cases of myeloma, 12 cases of megaloblastic anemia, eight cases of aplastic anemia, five cases of IAT and LLS, three cases of hypersplenism, two cases of bone marrow metastasis and one case of blast crisis of CML. Maximum number of cases (38 cases, 26.4%) was seen in less than ten years of age group followed by 18.7% cases (27 cases) in 21-30 years of age. Least number of cases was seen in more than 61 years of age (nine cases, 6.3%). Thrombocytopenia was commoner in males (24 cases, 63.2%) than in females (14 cases, 36.8%) in the first decade.
The changes in number and morphology of megakaryocytes in various hematological disorders are given in [Table 1] and [Table 2]. There was an increase in the number of megakaryocytes in 18 cases (94.7%) of ITP and immature megakaryocytes were seen in all the cases (sensitivity = 100%, specificity = 68%, [Figure 1]). Dysplastic forms were seen in 17 cases (89.5%), bare megakaryocytic nuclei in 16 cases (84.2%) and micromegakaryocytes in eight cases (42.1%) of ITP, their sensitivity being 89%, 84% and 42% respectively and specificity being 52%, 58% and 84% respectively. Emperipolesis was seen in 13 cases (68.4%; sensitivity = 68%, specificity = 74%) with lymphocytes in five cases and lymphocytes and nucleated red blood cells in four cases.
In cases of IAT, immature megakaryocytes were observed in all the five cases (sensitivity = 100%, specificity = 61%) and cytoplasmic vacuolization was seen in two of the cases (sensitivity = 40%, specificity = 86%).
The dysplastic forms were seen in megaloblastic anemia and dimorphic anemia and their sensitivity in megaloblastic anemia was 75% along with specificity of 49%. The sensitivity of emperipolesis in cases of dimorphic anemia was 44% [Figure 2]. However no platelet budding was seen in any of the cases.
In cases of MDS, dysplastic forms, bare megakaryocytic nuclei and micromegakaryocytes were seen. However, finding of micromegakaryocytes was most significant when compared to non-MDS causes of thrombocytopenia (specificity - 83%). Decreased platelet budding and absence of cytoplasmic vacuoles was also noted.
Thrombocytopenia, either persistent, isolated or in association with pancytopenia refractory to treatment is one of the commonly encountered hematological problems for which a bone marrow study is sought. The routinely prepared Leishman stained bone marrow aspirate smears can help to observe the dyspoietic features of the megakaryocytes associated with the non-MDS conditions. This can improve the diagnostic accuracy for a wide range of hematological disorders thereby enabling proper therapeutic interventions.
Normal maturation and platelet formation results from megakaryocytic deoxyribonucleic acid (DNA) replication that occurs without cell division resulting in large lobulated, polypoid nucleus. A wide variety of growth factors like thrombopoietin act synergistically with other hematopoietic cytokines and transcriptional factors stimulating the maturation and growth of megakaryocytes.  A defect in any of the stages of megakaryocytopoiesis can lead to dysmegakaryocytopoiesis and thrombocytopenia.
A shift to young, immature, less polypoid megakaryocytes and fewer mature platelet-producing megakaryocytes was the outstanding morphological feature noted in all the cases of ITP in the present study (sensitivity = 100%, specificity = 68%). Similar findings were observed by Houwerzijl et al,  wherein they said it is because of apoptotic and para-apoptotic type of programmed cell death (PCD) of mature megakaryocytes. Most of the abnormal megakaryocytes were surrounded by neutrophils and macrophages, some in the state of phagocytosis. Inappropriate PCD of mature megakaryocytes can disrupt platelet formation and apoptosis-like PCD (para-apoptosis) occurs in ITP. This finding is especially useful when some patients of MDS present with isolated thrombocytopenia, thus mimicking ITP. Dysplastic forms were seen in 17 cases (89.5%), bare megakaryocytic nuclei in 16 cases (84.2%) and micromegakaryocytes in eight cases (42.1%), their sensitivity being 89%, 84% and 42% respectively and specificity, 52%, 58% and 84% respectively.
Emperipolesis, seen in 13 of the 19 cases of ITP (84.2%; sensitivity = 68%, specificity = 74%) with lymphocytes in five and lymphocytes along with nucleated red blood cells in four cases, correlated with that of Roznan and Vives - Corrons.  The cytoplasmic vacuolization seen in nine cases (47.4%) which ultrastructurally represents mitochondrial swelling was also observed by Levine  and Houwerzijl et al,  and this reflects an increased megakaryocyte turnover and indicates degenerative changes such as those of apoptosis and para-apoptosis. Another plausible explanation for the cytoplasmic vacuolization is autophagy to maintain cell metabolism when there is increased metabolic demand and nutrition deficiency due to increased megakaryocytopoiesis or it might be a way of sequestration and degradation of specific pathogens such as immunoglobulins. All the above features suggested dysmegakaryocytopoiesis which largely contributes to diminished platelet production.
The antiplatelet autoantibodies to glycoprotein IIb/IIIa and Ib/IX seen in ITP interfere with platelet production and release by causing megakaryocyte destruction and abnormal maturation. ,,, The destruction of megakaryocytes is either by phagocytic cells or by activation of complement or by induction of apoptosis.  They also inhibit megakaryocyte colony formation and proplatelet formation, the defect being in the common erythroid-megakaryocyte stem cell. Therefore, altered megakaryocytic morphology and destruction resulting in defective platelet production and immune-mediated platelet destruction together contribute to thrombocytopenia.  The fact that some patients respond to thrombopoietin mimetics further supports the fact that suppression of platelet production is an important mechanism in some ITP patients. 
In 18 of the 19 cases of ITP, there was an increase in the number of megakaryocytes which was also observed by George, Harake and Raskob,  and Levine.  They attributed this to stimulation of the marrow megakaryocytes to synthesize platelets at an increased rate due to immune-mediated platelet destruction in the spleen and other reticuloendothelial tissues. The severity of thrombocytopenia with increased megakaryocyotpoiesis correlates with increased mean platelet volume (MPV) in patients with ITP. A low MPV is observed in myeloproliferative disorders, hypersplenism and thrombocytopenia associated with sepsis. 
All five cases of IAT had increased megakaryocytes as noted by Alter, Scanlon and Schechter.  According to them, the virus might directly damage the platelets or alter them to become antigenic, resulting in specific antiplatelet antibody formation. Alternatively, a virus-antivirus complex could precipitate on the platelets and damage them resulting in compensatory increase of megakaryocyte in the bone marrow.  Immature megakaryocytes were observed in all the five cases (sensitivity = 100%, specificity = 61%) similar to that of Meindersma and de Vries  who opined that this was due to the increased megakaryocyte turnover. Cytoplasmic vacuolization seen in two of the cases (sensitivity = 40%, specificity = 86%), correlated with that of Chanarin and Walford  and Chesney et al.  who pointed out that in acquired cytomegalovirus infection, this is due to toxic change caused by the virus. Recent studies have shown immune-mediated platelet destruction to be the cause of thrombocytopenia in human immunodeficiency virus, Hepatitis C virus and Helicobacter pylori infections. 
Megakaryocytes were decreased or absent in aplastic anemia which was also observed by Shadduck.  They attributed this to bone marrow suppression and significant inhibition of nucleic acid synthesis in the megakaryocytes. The hypolobated forms and dysplastic forms seen were in contrast to those of Tricot et al.  where megakaryocytes were of normal morphology. The dyspoietic megakaryocytes which were present in one case of aplastic anemia showed that the patient was a known case of ITP who had subsequently developed aplastic anemia and these abnormal megakaryocytes had persisted in the marrow. Presence of stem cell defect in ITP patients can progress to overt marrow failure.
In megaloblastic anemia, dysplastic forms were seen in nine cases (75%; sensitivity= 75%, specificity= 49%), which were contrary to the findings of Tricot et al.  However, Wickramasinghe  observed megakaryocytes with separation of nuclear lobes and nuclear fragments and attributed this to diminished DNA synthesis leading to nuclear maturation defect. The finding of emperipolesis in anemia was in agreement with the observation of Tavassoli.  However, no platelet budding was observed in any of the cases.
The increased number of megakaryocytes observed in all cases of hypersplenism is compensatory and is due to removal of platelets by increased pooling and by increased phagocytosis in the spleen. 
Three of the five cases (60%) of leukemia-lymphoma syndrome showed decreased or absent megakaryocytes. This may be because of the autoantibodies against glycoprotein IIa-IIIb complex which have been demonstrated in patients with lymphoma. According to Lim and Ifthikharuddin,  along with immune-mediated platelet destruction, decreased platelet production when the marrow is involved by lymphoma, bone marrow suppression by chemotherapeutic agents and platelet sequestration in the spleen also contribute to thrombocytopenia in lymphoma. There were 27 cases (18.8%) of AML in the present study and this was the most common cause of thrombocytopenia for which bone marrow examination was sought. In 17 of the cases (63%), the number of megakaryocytes was decreased with six cases (22.2%) not showing any megakaryocytes. Tricot et al,  reported the same in marrows of AML tightly packed with leukemic blast cells with maturation arrest. The immature forms, dysplastic forms, bare megakaryocyte nuclei and emperipolesis were observed in cases of leukemias and LLS. The exact mechanism is not clear due to paucity of literature in this aspect. The five cases (26.3%) of AML which also showed micromegakaryocytes might be considered as a transformation from MDS. Further studies are required to sufficiently explain the dyspoietic features in these neoplastic conditions.
Dyspoietic megakaryocytes are well described in MDS, however, limited literature exists regarding their significance in non-MDS cases. In the present study, there was no significant difference in the dyspoietic features in non-MDS cases as compared to MDS cases except for the micromegakaryocytes (P  Hence, dyspoietic features by themselves do not specify MDS, other hematological conditions causing thrombocytopenia have to be considered in the differential diagnosis. However, this study included only a few cases of MDS for comparison. Studies with increased sample size should be done for further evaluating their significance. Various dyspoietic features were observed in all the non-MDS cases, of which there was significant difference in the presence of micromegakaryocytes between cases of MDS and non-MDS.
To summarize, the sensitivity of dysplastic forms and micromegakaryocytes in cases of ITP was 89% and 42% respectively and specificity 52% and 84% respectively. In cases of infection-associated thrombocytopenia, immature megakaryocytes had sensitivity of 100% and cytoplasmic vacuolization was 86% specific. The sensitivity of the dysplastic forms in megaloblastic anemia was 75%. However, no platelet budding was observed. There was no significant difference in the dyspoietic features in non-MDS cases as compared to MDS cases except for the micromegakaryocytes (P<0.05) with specificity of 83% in MDS. Decrease in platelet budding and absence of cytoplasmic vacuoles was also noted in cases of MDS. The observed megakaryocytic alterations can be made useful in routine reporting of bone marrow aspirations on non-MDS related thrombocytopenias.
Further studies on the evaluation of megakaryocytic alteration and their contribution to thrombocytopenia can provide growing knowledge to the pathogenesis of numerous hematopoietic disorders that may identify broader clinical applications of the newer strategies to regulate platelet count and functioning.
|1||McKenzie SB, editor. Textbook of hematology. 2nd ed. Pennsylvania: Willaims and Wilkins; 1996.|
|2||Houwerijl EJ, Blom NR, van der Want JJ, Esselink MT, Koornstra JJ, Smit JW, et al. Ultrastructural study shows morphologic features of apoptosis and para-apoptosis in megakaryocytes from patients with idiopathic thrombocytopenic purpura. Blood 2004;103:500-6.|
|3||Battinelli EM, Hartwig JH, Italiano JE Jr. Delivering new insight into the biology of megakaryopoiesis and thrombopoiesis. Curr Opin Hematol 2007;14:419-26. |
|4||Rozman C, Vives-Corrons JL. On the alleged diagnostic significance of megakaryocytic 'phagocytosis' (emperipolesis). Br J Haematol 1981;48:510.|
|5||Levine FC. "Idiopathic" thrombocytopenia. Arch Intern Med 1999;88:701-28.|
|6||Wang L, Li Y, Hou M. Idiopathic thrombocytopenic purpura and dysmegakaryocytopoiesis. Crit Rev Oncol Hematol 2007;64:83-9.|
|7||McMillan R, Wang L, Tomer A, Nicho J, Pistello J. Suppression of in vitro megakaryocyte production by antiplatelet autoantibodies from adult patients with chronic ITP. Blood 2004;103:1364-9.|
|8||McMillan R. The pathogenesis of chronic immune thrombocytopenic purpura. Semin Hematol 2007;44:S3-S11. |
|9||George JN, el-Harake MA, Raskob GE. Chronic idiopathic thrombocytopenic purpura. N Engl J Med 1994;331:1207-11. |
|10||Wang ZY, Shen ZX. Megakaryocytes and platelets in immune thrombocytopenic purpura. Baillieres Clin Haematol 1997;10:89-107.|
|11||Alter JH, Scanlon RT, Schechter PG. Thrombocytopenic purpura following vaccination with attenuated measles virus. Am J Dis Child 1969;115:111-6.|
|12||Meindersma TC, de Vires SI. Thrombocytopenic purpura after small pox vaccination. Br Med J 1962;1:226-9.|
|13||Chanarin I, Walford DM. Thrombocytopenic purpura in cytomegalovirus mononucleosis. Lancet 1973;2:238-9.|
|14||Chesney PJ, Taher A, Gilbert EM, Shahadi NT. Intranuclear inclusions in megakaryocytes in congenital cytomegalovirus infection. J Pediatr 1978;92:957-60. |
|15||Liebman H. Other immune thrombocytopenias. Semin Hematol 2007;44:S24-34.|
|16||Shadduck. Aplastic anemia: review of 27 cases. Lancet 2001;1:657-67.|
|17||Tricot G, Vlietinck R, Boogaerts MA, Hendrickx B, De Wolf-Peeters C, Van den Berghe H et al. Prognostic factors in the myelodysplastic syndromes: importance of initial data on peripheral blood counts, bone marrow cytology, trephine biopsy and chromosomal analysis. Br J Haematol 1985;60:19-32.|
|18||Wickramasinghe SN. Morphology, biology and biochemistry of cobalamine- and folate- deficient bone marrow cells. Baillieres Clin Haematol 1995;8:441-59.|
|19||Tavassoli M. Modulation of megakaryocyte emperipolesis by phlebotomy: megakaryocytes as a component of marrow-blood barrier. Blood cells 1986;12:205-16. |
|20||Diz-Kucukkaya R, Gushiken FC, Lopez JA. Thrombocytopenia. In: Lichtman MA, Beutler E, Kipps T, Seligsohn U, Kaushansky K. Prchal JT, editors. Williams Haematology. 7th ed. USA: McGraw-Hill; 2006. p. 1749-83. |
|21||Lim SH, Ifhtikharuddin JJ. Autoimmune thrombocytopenic purpura complicating lymphoproliferative disorders. Leuk Lymphoma 1994;15:61-4. |
|22||Houwerzijl EJ, Blom NR, van der Want JJL, Vallenga E, de Wolf JTM. Megakaryocytic dysfunction in myelodysplastic syndromes and idiopathic thrombocytopenic purpura is in part due to different forms of cell death. Leukemia 2006;20:1937-42.|