|Year : 2017 | Volume
| Issue : 1 | Page : 51-57
A study of megakaryocyte morphology in bone marrow aspiration smears of cases of thrombocytopenia
Shashikala Vinayakamurthy, Rojaramani Potluri, Prathima Shivajirao, Rekha Singh, Rajalaxmi Pujahari, Indu Maniketh
Department of Pathology, Vydehi Institute of Medical Sciences and Research Centre, Bengaluru, Karnataka, India
|Date of Web Publication||9-Jan-2017|
Dr. Shashikala Vinayakamurthy
Vydehi Institute of Medical Sciences and Research Centre, Bengaluru - 560 066, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Thrombocytopenia may be encountered in various hematological and nonhematological conditions and may be associated with dysplastic megakaryocytes which is a feature of myelodysplastic syndrome (MDS), even though they can be observed in non-MDS hematological conditions. Objective: To study the morphological variations of megakaryocytes on bone marrow aspiration smears in non-MDS-related thrombocytopenia in a Medical College in Bengaluru, Karnataka. Materials and Methods: It was a prospective study of 86 cases of non-MDS thrombocytopenia whose bone marrow aspirates were studied morphologically. Results: The most common cause of thrombocytopenia was acute leukemia followed by other systemic malignancies, megaloblastic anemia, and idiopathic thrombocytopenic purpura. Both dysplastic and nondysplastic features were observed in the above-mentioned conditions. The most common dysplastic feature was nuclear segmentation followed by micromegakaryocytes and hypogranular forms. Among nondysplastic features, the most common were immature forms, bare nuclei, and hypolobation. Emperipolesis and cytoplasmic vacuoles were noted in a case of pyrexia of unknown origin. Conclusion: Dysplastic megakaryocytes are common in non-MDS-related thrombocytopenia and their mere presence should not lead to the diagnosis of MDS. Hence, proper diagnosis should be made on megakaryocyte morphology, patient's clinical findings, and other hematological parameters. This understanding can improve the diagnostic accuracy for wide range of hematological disorders.
Keywords: Bone marrow aspiration, dysplastic features, nonmyelodysplastic syndrome, thrombocytopenia
|How to cite this article:|
Vinayakamurthy S, Potluri R, Shivajirao P, Singh R, Pujahari R, Maniketh I. A study of megakaryocyte morphology in bone marrow aspiration smears of cases of thrombocytopenia. Med J DY Patil Univ 2017;10:51-7
|How to cite this URL:|
Vinayakamurthy S, Potluri R, Shivajirao P, Singh R, Pujahari R, Maniketh I. A study of megakaryocyte morphology in bone marrow aspiration smears of cases of thrombocytopenia. Med J DY Patil Univ [serial online] 2017 [cited 2020 Apr 2];10:51-7. Available from: http://www.mjdrdypu.org/text.asp?2017/10/1/51/197922
| Introduction|| |
Thrombocytopenia characterized by platelets count <150,000/µl is a common indication for which bone marrow aspiration is often performed. There are hematological and nonhematological causes. The most common hematological conditions for thrombocytopenia are megaloblastic anemia, aplastic anemia, leukemia, multiple myeloma, bone marrow metastasis, etc., Thrombocytopenia can be isolated or may be associated with bicytopenia or pancytopenia.
Dysplastic changes are commonly seen in patient with thrombocytopenia, especially associated with myelodysplastic syndrome (MDS). However, dysplastic changes of megakaryocytes may also be observed in non-MDS hematological condition. This study was to determine the morphology of megakaryocytes in a patient with non-MDS thrombocytopenia.
| Materials and Methods|| |
This was a prospective study of 86 cases of bone marrow aspirate (BMA) of patients presenting with platelet count <150,000/mm 3 from June 2014 to August 2015 at Vydehi Institute of Medical Sciences and Research Centre, Bengaluru, Karnataka. Written informed consent was obtained from all patients. Relevant clinical data were obtained using a pro forma. The automated platelet count was further confirmed by manual platelet count by preparing a peripheral smear and staining with Leishman's stain. The blood films were prepared for all cases to look for any evidence of pseudothrombocytopenia such as platelet agglutination, satellitosis, and phagocytosis by other cells. The distributions of morphological alterations in megakaryocytes were studied, and sensitivity and specificity of each feature were calculated using the Epi info is a free software package developed by the United states of America centers for disease control.
Cases with pseudothrombocytopenia and with inadequate material on bone marrow aspiration were excluded from the study. Bone marrow aspiration was done under standard aseptic conditions and the bone marrow smears were stained with Leishman's stain and examined. Leishman's stain was preferred in the study as it is a simple and rapid method and also provides better staining and contrast to the nuclear structure.
The BMA smears were examined as per the standard guidelines and the findings were documented. The number and morphology of the megakaryocytes were studied. The number of megakaryocytes was expressed as number per 10 low-power field (LPF) and was further subdivided into absent, decreased (1/5–10 LPF), normal (1/1–3 LPF), and increased (>2/LPF).,,
The morphological changes of megakaryocytes that were studied include both dysplastic and nondysplastic features. Dysplastic features included multiple separated nuclei, micro megakaryocytes, and hypogranular forms. Micromegakaryocytes were those megakaryocytes with a size similar to that of large lymphocytes or monocytes and had single or bilobed nuclei. Hypogranular forms were those megakaryocytes which had a clear cytoplasm with no or sparse granules. Nondysplastic features included immature forms, emperipolesis, platelet budding, cytoplasmic vacuolization, and bare megakaryocyte nuclei. Immature forms are the young megakaryocytes which lack nuclear lobulation and have scant basophilic cytoplasm.
At least thirty megakaryocytes were evaluated on BMA smears, and dysplastic alterations were considered significant only when 10% or more of megakaryocytes observed show the changes.
| Results|| |
Thrombocytopenia was more common in males with a male: female ratio of 2.5:1. Thrombocytopenia was the most common in age group of 30–39 years (22.09%) as shown in [Table 1]. The least number of cases were in the age group of 70–89 years.
The causes of thrombocytopenia are shown in [Table 2]. The most common cause for thrombocytopenia was acute leukemia.
There was an increase in number of megakaryocytes in BMA smears in cases of idiopathic thrombocytopenic purpura (ITP), systemic malignancies, metastatic deposits to bone marrow, megaloblastic anemia, and iron-deficiency anemia with sensitivity of 80%, 68.78%, 50%, 60%, and 62.5% and specificity of 60%, 62.66%, 48%, 61%, and 58%, respectively, as shown in [Table 3]. Decreased numbers of megakaryocytes were seen in acute leukemia. Megakaryocytes were absent in two cases of ITP and a case of aplastic anemia.
|Table 3: Distribution of megakaryocytes in various haematological disorders with thrombocytopenia|
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Both dysplastic and nondysplastic morphological features of megakaryocytes were studied as shown in [Table 4] and [Table 5]. The most common dysplastic features encountered were multiple segmented nuclei and micromegakaryocytes. Nuclear segmentation was noted in 8 cases of ITP (sensitivity of 80% and specificity of 50%) and 12 cases of megaloblastic anemia (sensitivity of 80% and specificity of 52%), followed by 12 cases of acute leukemia (sensitivity of 60% and specificity of 52%), 1 case of aplastic anemia (sensitivity of 50% and specificity of 45%), and 8 cases of other systemic malignancies without bone marrow involvement (sensitivity and specificity of 50%). Micromegakaryocytes were encountered in 7 cases of ITP (sensitivity of 70% and specificity of 72%) and 1 case of aplastic anemia (Sensitivity of 50% and specificity of 67%). Hypogranular forms were seen in 6 cases of ITP with a sensitivity of 60% and specificity of 91%.
|Table 4: The occurrence of dysplastic features of megakaryocytes in various haematological disorders along with their sensitivity of diagnosis|
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|Table 5: The occurrence of non dysplastic features of megakaryocytes in various haematological disorders along with their sensitivity of diagnosis|
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Among nondysplastic features, the most common were immature forms, emperipolesis, cytoplasmic budding, bare nuclei, and hypogranulations.
Immature megakaryocytes were seen in 8 cases of ITP (sensitivity of 80% and specificity of 51%), 11 cases of megaloblastic anemia (sensitivity of 73% and specificity of 53%), 10 cases of other systemic malignancies without marrow involvement (sensitivity of 62% and specificity of 52%), and 9 cases of acute leukemia (sensitivity of 45% and specificity of 51%). Emperipolesis was observed in 4 cases of ITP (sensitivity of 60% and specificity of 89%) and a case of pyrexia of unknown origin (PUO) (sensitivity of 100%). Cytoplasmic budding on the surface of megakaryocytes was noted in 6 cases of megaloblastic anemia (sensitivity of 40% and specificity of 78%), 4 cases of ITP (sensitivity of 40% and specificity of 76%), 5 cases of systemic malignancies (sensitivity of 35% and specificity of 77%), and a case of aplastic anemia (sensitivity of 50% and specificity of 74%). Bare nuclei were another common feature encountered in 14 cases of megaloblastic anemia (sensitivity of 93% and specificity of 65%), 5 cases of ITP (sensitivity of 50% and specificity of 58%), and 8 cases of acute leukemia (sensitivity of 40%% and specificity of 60%). They were also seen in cases of ILD and PUO. Hypolobated forms were seen in 10 cases of megaloblastic anemia (sensitivity of 66% and specificity of 71%), 6 cases of ITP (sensitivity of 60% and specificity of 67%), and 4 cases of systemic malignancy (sensitivity of 56% and specificity of 65%).
| Discussion|| |
Of the 86 cases of thrombocytopenia studied, the most common cause was acute leukemia (23.25%), followed by other systemic malignancies (18.60%), megaloblastic anemia (17.44%) and ITP (11.60%). Our findings are comparable with the study done by Muhury et al., where the most common cause for thrombocytopenia was acute myeloid leukemia (AML) (18.8%), followed by ITP (13.5%) and ALL (12.5%). The most common age group in our study was 30–39 years followed by 0–9 years. Our institution has a well-established oncology center and caters to wide range of malignancies including pediatric tumors. This could be the reason for increased number of cases in the above age group. In our study, thrombocytopenia was the most common in males, 72.09%. This was similar to that observed by Muhury et al. and by Parul et al.
We found that 80% cases of ITP showed increased number of megakaryocytes in BMA smears as shown in [Figure 1], the average being 64/10 LPF. This corresponds to the study done by Choudhary et al., Shi et al., and Muhury et al., where they found an increase in megakaryocytes in 91%, 98%, and 95% cases of ITP, respectively. Jubelirer et al. also had similar findings with 95.3% cases showing an increase in megakaryocytes in cases of ITP. This could be due to stimulation of the marrow megakaryocytes to synthesize platelets at an increased rate due to their immune-mediated destruction in the spleen and other reticuloendothelial tissues.
|Figure 1: Microphotograph displaying increased megakaryocytes in a case of idiopathic thrombocytopenic purpura (Leishman, ×4)|
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Increase in megakaryocytes was seen in malignancies (68%) and in metastatic deposits (50%) to bone marrow. Megakaryocytes ploidy was found to be significantly higher in patients with metastatic disease, thus leading to increased production and altered platelet heterogeneity. Studies have shown that interleukin-6 is elevated in a variety of malignancies and acts as thrombopoietic agent.,,
Sixty percent of cases of megaloblastic anemia showed an increase in a number of megakaryocytes. This corresponds to a study done by Parul et al., where 58.3% cases showed an increase in a number of megakaryocytes.
Megakaryocytes were also found to be increased in 62.5% cases of iron-deficiency anemia (IDA). This could be explained by the experimental study done on rat which showed hypoxia-induced factors 1, 2 sub-unit (HIF-22) and vascular endothelial growth factor to be increased in rats and cultural supernatants.
Our study showed that 65% cases of acute leukemia had decreased number of megakaryocytes. This could be due to leukemic cells infiltrating the marrow leading to decreased production. Other factors for reduced megakaryocytes include chemotherapy, radiotherapy, and immune-mediated destruction of megakaryocytes.
This study found that 20% cases of ITP had absent megakaryocytes without marrow hypocellularity and was diagnosed as amegakaryocytic thrombocytopenia. No cytogenetic studies were done in these cases as the patient refused for further workup. Other condition, where megakaryocytes were absent, was in a case of aplastic anemia, similar to the observations by Shadduk RKet al.
In our study, dysplastic megakaryocytes were appreciated in megaloblastic anemia, ITP, and in cases of acute leukemia. The most common dysplastic feature was multiple segmented nuclei seen in 80% cases of megaloblastic anemia and ITP and 60% cases of acute leukemia. This is attributed to diminished DNA synthesis and increased ploidy leading to nuclear maturation defect.
Of ten cases of ITP, 80% of cases showed nuclear segmentation as shown in [Figure 2], 70% showed micro megakaryocytes, and 60% of cases had hypogranular forms. These dysplastic features are attributed to the presence of IgG autoantibodies which bind to the Glycoprotein II b/IIIa and Glycoprotein Ib/Ix expressed on the surface of the committed megakaryocyte progenitors and have a deleterious effect on megakaryopoiesis. This study corresponds to Murthy et al., where he found dysplastic features in 89.5% cases. He found multiple segmented nuclei in 15.2% cases and micro megakaryocytes in 61% cases. Wickramasinghe also found that the most common dysplastic feature was multiple separated nuclei and attributed it to diminished and ineffective DNA synthesis leading to maturation defect.
|Figure 2: Microphotograph displaying multiple segmented nuclei in a case of idiopathic thrombocytopenic purpura (Leishman, ×100)|
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Among the nondysplastic features, immature and hypolobulated forms were the most common as shown in [Figure 3]. This is attributed to the programmed cell death of mature megakaryocytes seen in ITP. This finding is useful in patients with MDS, who present with isolated thrombocytopenia, thus mimicking ITP. Other common findings in ITP cases were bare nuclei seen in 70% of cases and emperipolesis in 40% of cases. This finding corresponds to Muhury et al., who found 84.2% cases with bare nuclei and 68.4% cases with emperipolesis in ITP cases. Rai et al. also found 68.4% cases of ITP exhibiting emperipolesis. Tavassoli et al. attributed this to cells taking a transmegakaryocytic route to enter the circulation causing the phenomenon of emperipolesis and thus suggested megakaryocytes as a component of the marrow-blood barrier.
|Figure 3: Microphotograph displaying immature megakaryocytes in a case of idiopathic thrombocytopenic purpura (Leishman, ×4)|
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Our study also showed many cases of megaloblastic anemia displaying dysplastic features. Eighty percent of cases showed nuclear segmentation and 33% cases showed micromegakaryocytes. This is attributed to diminished DNA synthesis leading to nuclear maturation defect. Wickramasinghe also found that the most common dysplastic feature was multiple segmented nuclei and attributed it to diminished and ineffective DNA synthesis leading to maturation defect. Ashalatha et al. found 100% cases of megaloblastic anemia with dysplastic features.
Among nondysplastic features, the most common in megaloblastic anemia were immature forms (73.3%), bare nuclei (93.3%), hypolobated forms (66%), nuclear budding (40%), and emperipolesis (26%). The mechanism for such nondysplastic features according to Wang et al. is due to impaired DNA synthesis and methylation due to folate and Vitamin B12 deficiency.
Dysplastic features were also documented in cases of acute leukemia. Sixty percent of cases showed nuclear segmentation, 40% showed micro megakaryocytes, and 10% showed hypogranular forms. Lee et al. found that 59.4% of cases of acute leukemia showed micromegakaryocytes. He also found that these patients were associated with bad prognosis.
Nondysplastic features commonly encountered in cases of acute leukemia were immature forms (45%), hypolobation (40%), bare nuclei (40%), emperipolesis (5%), and budding (10%). Brandt et al. had found, using banding technique that in AML, an isochromosome 17 was found in granulopoietic, erythropoietic cells, and megakaryocytes. He also found that most megakaryocytes were diploid and the polyploidy was not normally developed. Polyploidy results in defective differentiation of the megakaryocytes and their precursors.
Megakaryocytic dysplasia was also seen in cases of other systemic malignancies without bone marrow involvement. In our study, we had 16 cases of other systemic malignancies comprising Hodgkin's and non-Hodgkin's lymphomas, lung malignancies, gastrointestinal malignancies, and prostatic malignancies.
We had eight cases of iron-deficiency anemia and 25% of cases showed multiple segmented nuclei and micromegakaryocytes. Evstatiev et al. have studied the effect of iron deficiency on megakaryopoiesis in experimental animals and found that iron deficiency inhibits proliferation but increases ploidy in megakaryocytic cell lines. This may affect the normal maturation process.
Among the nondysplastic features in IDA, immature forms, bare nuclei, and hypolobated forms were noted.
In our study, we had two cases of aplastic anemia in which one of the cases showed absent megakaryocytes and the other cases showed nuclear segmentation and micro megakaryocytes. Parul et al. found 100% cases of aplastic anemia showing features of micro megakaryocytes and hypogranular forms.
Other cases in our study were one each of PUO, interstitial lung disease, and cirrhosis of the liver. These cases did not show any dysplastic features. However, nondysplastic features were encountered in them such as emperipolesis, cytoplasmic vacuolization, nuclear budding, and bare nuclei.
PUO showed the evidence of cytoplasmic vacuolization as shown in [Figure 4] and emperipolesis. Chanarin and Walford and Chesney et al. pointed out that these findings were due to toxic change caused by the infectious agents.,
|Figure 4: Microphotograph displaying cytoplasmic vacuolation in a case of pyrexia of unknown origin (Leishman, ×4)|
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One case of MDS was studied which showed both dysplastic and nondysplastic features such as nuclear segmentation, immature forms, hypo granular forms, and nuclear budding.
Dysplastic features represent megakaryocytes that are abnormal and have lost their ability to undergo proper differentiation and have impaired nuclear development. We found dysplastic features in condition with thrombocytopenia such as megaloblastic anemia, ITP, leukemias, and other systemic malignancies. We also want to highlight in our study that dysplastic features with or without thrombocytopenia can also be noted in IDA. Further studies have to be carried out regarding IDA association with dysplastic features.
Hence, this study shows that dysplastic megakaryocytes are also found in non-MDS-related thrombocytopenia and their existence alone does not qualify for the diagnosis of MDS and other hematological conditions to be considered in the differential diagnosis.
| Conclusion|| |
The most common cause of non-MDS thrombocytopenia in our study was acute leukemias, megaloblastic anemia, other malignancies, ITP, and IDA. Dysplastic features were observed in all these conditions and more commonly in cases of megaloblastic anemia ITP and acute leukemias. Hence, we conclude that mere presence of dysplastic features should not lead to diagnosis of MDS, but other differential diagnoses be considered.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]