Original article / research
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Bone Marrow Cellularity in Pancytopenia- A Paradigm of Underlying Pathology |
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Alok Kumar, Parineeta Shelke, Preeti Rajeev Doshi, Amit Nisal, Ravindra Nimbargi 1. Postgraduate Student, Department of Pathology, Bharati Vidyapeeth (DTU) Medical College, Pune, Maharashtra, India. 2. Assistant Professor, Department of Pathology, Bharati Vidyapeeth (DTU) Medical College, Pune, Maharashtra, India. 3. Associate Professor, Department of Pathology, Bharati Vidyapeeth (DTU) Medical College, Pune, Maharashtra, India. 4. Associate Professor, Department of Pathology, Bharati Vidyapeeth (DTU) Medical College, Pune, Maharashtra, India. 5. Professor, Department of Pathology, Bharati Vidyapeeth (DTU) Medical College, Pune, Maharashtra, India. |
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Correspondence
Address : Preeti Rajeev Doshi, Associate Professor, Department of Pathology, Bharati Vidyapeeth (Deemed to be University) Medical College, Pune, Maharashtra, India. E-mail: prdoshi22@gmail.com |
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ABSTRACT | |||||||||||||||||||||||||||||||||||||||||||||||||||||
: Reduced numbers of all three types of peripheral blood cells characterise the haematologic condition known as pancytopenia. Practical distinction among various causes of pancytopenia is usually clear but some processes are so closely related that the diagnosis may get complicated and bone marrow examination aids in diagnosis of such cases. It is important to recognise marrow failure syndromes causing pancytopenia. Pancytopenia is a common finding, its explicit discussion is lacking even in major textbooks and has led many authors to highlight the spectrum of causes of pancytopenia. Aim: To evaluate the various causes of pancytopenia and to evaluate clinical signs and symptoms, haematological parameters along with bone marrow cellularity and other morphological features on aspiration and trephine biopsy in patients presenting with pancytopenia. Materials and Methods: This cross-sectional study was conducted for a period of 3.5 years from August 2018 to April 2022, on total 157 patients having pancytopenia in a tertiary care centre, Pune, Maharashtra, India. Clinical history was taken for all the cases of pancytopenia. The blood samples were collected for haematological analysis including haemogram and Peripheral Blood Smear (PBS) examination; also Bone Marrow (BM) samples were collected. Aspirates were stained with Leishman and Giemsa. Special stains like Myeloperoxidase (MPO) and Periodic Acid Schiff stain (PAS) were used wherever required. Bone marrow biopsy was fixed in Bouin’s fluid; processed and stained with Haematoxylin and Eosin (H&E) and reticulin stain after decalcification. Results were analysed using Statistical Package for the Social Sciences (SPSS) software (version 26.0) and calculated as frequencies and percentages. A p-value ≤0.05 was considered significant. Results: Out of 157 patients, majority 120 (76.43%) belonged to adult age group (18-86 years), with the mean age of 40.68±23.34 years. The male to female ratio was 1.34:1. Study showed megaloblastic anaemia encompassing majority of the causes of pancytopenia followed by acute leukaemia, hypersplenism, hypocellular marrow, Haemophagocytic Lymphohistiocytosis (HLH), Myelodysplastic Syndrome (MDS) and aplastic anaemia. Out of 86 (54.78%) of total majority of hypercellular bone marrow patients, 51 (59.3%) had haemoglobin levels of <7 g/dL, while 45 (52.32%) hypercellular bone marrow patients had platelet count of <50000 cells/cumm. Patients with low TLC were significantly associated with hypocellular (p=0.0067) and hypercellular marrow (p=0.0291) compared to normocellular marrow. For reticulocyte count, an increasing trend with low reticulocyte count was seen from normocellular (n=4, 6.9%) to hypocellular (n=12, 20.7%) to hypercellular (n=19, 32.8%) bone marrow, though it was not statistically significant. Conclusion: It was concluded from the present study that megaloblastic anaemia was the most common aetiology of pancytopenia and the most common clinical symptoms observed was fever. | |||||||||||||||||||||||||||||||||||||||||||||||||||||
Keywords : Bone marrow evaluation, Haemophagocytic lymphohistiocytosis, Megaloblastic anaemia, Pancytopenia | |||||||||||||||||||||||||||||||||||||||||||||||||||||
INTRODUCTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||
In daily clinical work, pancytopenia is a frequent finding. There is a lack of red blood cells, white blood cells, and platelets in the blood. Symptoms include a haemoglobin level below 12 g/dL in women and 13 g/dL in men, platelet count below 150,000/mcL, and white blood cell count below 4000/mL (or absolute neutrophil count of less than 1800 per mL) (1),(2). The incidence of pancytopenia peaks in the third and fourth decades of life for both children and adults. According to the published data, the male to female ratio ranges from 1.4% to 2.6%. Myelodysplastic syndrome and multiple myeloma are more common in older patients, while acute leukaemia and parvovirus B19 infection are more common in younger patients (3). There are many potential causes of pancytopenia, and diagnosing the underlying condition can be difficult due to the breadth of possible causes. Pancytopenia, a mild impairment in marrow function, may not be noticed until times of stress or increased demand, (4). Pancytopenia is a symptom of many different diseases, both haematopoietic and otherwise. There are a number of underlying mechanisms at play, including a decrease in haematopoietic cell production, the replacement of marrow with abnormal cells, the suppression of marrow growth and differentiation, ineffective haematopoiesis with cell death, defective cell formation that results in the removal of cells from circulation, antibody-mediated sequestration or destruction of cells, and the trapping of cells in a hypertrophied and overactive reticuloendothelial system (1),(5),(6). Hypersplenism, infections, myelosuppression (cancer, chemotherapy, drug toxicity, or radiotherapy), and megaloblastic anaemia were found to be the most common causes in a 2013 study conducted in India (7). Megaloblastic anaemia was found to be most common cause of pancytopenia followed by combined nutritional deficiency, aplastic anaemia, iron deficiency anaemia, acute leukaemia, Non Hodgkin Lymphoma (NHL), Juvenile Myelomonocytic Leukaemia (JMML), Myelodysplastic Syndrome (MDS), Primary Myelofibrosis (PMF), and storage disorder in another study conducted in India in 2022 (8). Megaloblastic anaemia was found to be the leading cause of anaemia in India, with aplastic anaemia coming in second (9). As the principle site of haematopoiesis is BM, low blood counts necessitates the BM examination. Practical distinction among various causes of pancytopenia is usually clear but some processes are so closely related that the diagnosis may get complicated and BM examination aids in diagnosis of such cases. It is important to recognise marrow failure syndromes causing pancytopenia in order to improve the prognosis of patients (10),(11). Although pancytopenia is a common finding, its explicit discussion is lacking even in major textbooks and has led many authors to highlight the spectrum of causes of pancytopenia. To better understand the causes of pancytopenia and develop tactics to address them, the current cross-sectional study was designed.The present study was carried out to evaluate the various causes of pancytopenia and to evaluate clinical signs and symptoms, haematological parameters along with BM cellularity and other morphological features on aspiration and trephine biopsy in patients presenting with pancytopenia. | |||||||||||||||||||||||||||||||||||||||||||||||||||||
MATERIAL AND METHODS | |||||||||||||||||||||||||||||||||||||||||||||||||||||
This cross-sectional study was done in Department of Pathology, Bharati Vidyapeeth (Deemed to be University) Medical College, Pune, Maharashtra, India from August 2018 to April 2022. Total 157 patients presenting with pancytopenia were recruited from a tertiary care hospital in western part of state of Maharashtra, India. The study was approved by the Institutional Ethics Committee, BVDUMC/IEC/67. Sample size calculation: The sample size was calculated to be 148 at 95% confidence interval and 8% of margin of error considering the prevalence of 43% (12). Authors recruited additional 9 patients to make total of 157. Inclusion criteria: All patients with pancytopenia were included in the study. Exclusion criteria: Patients who received chemotherapy/radiotherapy and dry tap were excluded from the study. Required clinical details were taken for all the cases. Procedure After informed consent was obtained from each participant included in the study, blood samples were collected in anticoagulated {K2 Ethylene Diamine Tetraacetic Acid (EDTA)} vacutainers for haemogram, the analysis of haemogram was carried out on fully automated analyser DXH 800 (Beckman Coulter) along with peripheral smear examination. Bone marrow aspiration was done from posterior superior iliac spine in all the patients with Salah needle, the aspirate was drawn with a 20 mL plastic syringe. Bone marrow smears were prepared immediately following aspiration. After being air dried, these smears where stained with Leishman and Giemsa for morphological examination. Special stains like Iron stain, Myeloperoxidase (MPO) and Periodic acid Schiff (PAS) stains were used, wherever necessary. Bone marrow biopsy was fixed in Bouin’s fluid and processed and stained with H&E and reticulin stain after decalcification, and then BM aspiration smears and biopsy sections were interpreted after thorough microscopic examination.Anaemia was classified as mild, moderate or severe based on the concentrations of haemoglobin in the blood. • Mild: ≥10.0 g/dL; • Moderate: 7.1-9.9 g/dL; • Severe anaemia: ≤7.0 g/dL (13). Statistical Analysis The data was analysed using SPSS software (version 26.0). The results of qualitative parameters were calculated as frequencies and percentages and compared between the groups using Chi-square test. The continuous variables were calculated as mean±SD, and compared between the means using students ‘t’ test. A p-value of <0.05 was considered significant. | |||||||||||||||||||||||||||||||||||||||||||||||||||||
RESULTS | |||||||||||||||||||||||||||||||||||||||||||||||||||||
Total 157 patients with pancytopenia were included in the study. Out of total, 120 (76.43%) patients belonged to adult age group (18-86 years) with mean age of the patients was 40.68±23.34 years. There were 67 (42.68%) females and 90 (57.32%) males as shown in (Table/Fig 1), with male:female ratio of 1.34:1. The most common clinical symptoms observed were fever 49 (31.20%), followed by generalised weakness 41 (26.11%), pallor 32 (20.40%), bleeding 14 (8.9%) and others as shown in (Table/Fig 2). Among the patients with pancytopenia, splenomegaly was predominant finding observed in 51 (32.5%) patients, followed by hepatomegaly in 31 (19.7%), hepatosplenomegaly in 16 (10.2%) (Table/Fig 3). Out of total 157 patients, data of organomegaly for 3 patients was not available due to lack of clinical history. Most common aetiology reported was megaloblastic anaemia in 35 (22.29%), followed by acute leukaemia in 29 (18.47%), hypersplenism and hypocellular marrow in 21 each (13.38% each), MDS 14 (8.92%), aplastic anaemia 04 (2.55%) and other aetiologies in 09 (5.73%) patients. The distribution of aetiologies as per BM analysis is shown in (Table/Fig 4). Most of the patients (n=88) had haemoglobin level of ≤7 g/dL, followed by level between 7.1 to 9.9 g/dL in 64 patients and there were only 5 patients with Hb level of ≥10 g/dL. Majority of the patients (n=113) had TLC values between 1000-3500/cu.mm, 27 patients had value of ≤1000/cu mm and only 17 had TLC of ≥3500/cu mm. Similarly, only 20 patients had platelet level of >100000/cu mm, while rest had platelet level of <100000/cu mm. Out of 157 patients, the reticulocyte count was carried out in 58 patients. Out of these 58 patients, 35 patients showed reticulocyte count less than 0.5% while 8 patients had reticulocyte count more than 2.5%. The BM aspirate in the present study of pancytopenia showed normocellular in 28 (17.83%), hypocellular 43 (27.39%) and hypercellular in 86 (54.78%) patients. The findings are depicted in (Table/Fig 5). The numbers of patients based on levels of haematological parameters were distributed among the normocellular, hypocellular and hypercellular on BM aspiration. There was a significant p-value (p=0.0067, p=0.0291) for total leucocyte count in hypocellular and hypercellular marrow aspirate sample respectively. The distribution of patients according to haematological parameters among normocellular, hypocellular and hypercellular BM samples is shown in (Table/Fig 6). The characteristic microscopic features of various causes of pancytopenia on BM examination are depicted in (Table/Fig 7). The BM aspiration showed hypercellular marrow fragments with trilineagehaematopoiesis in patients with hypersplenism (Table/Fig 7)a. In megaloblastic anaemia, BM aspiration showed megaloblastic erythroid hyperplasia. Megaloblasts had the characteristic feature of sieved nuclear chromatin, asynchronous nuclear maturation and bluish cytoplasm (Table/Fig 7)b. In present study acute lymphoid leukaemia was seen in 15 patients which showed L1 type lymphoblasts showing high N:C ratio, fine chromatin, inconspicuous nucleoli and scanty amount of pale blue cytoplasm (Table/Fig 7)c while patients with acute myeloid leukaemia showed predominantly large blasts with high nucleus: cytoplasmic ratio, fine chromatin and 1-2 prominent nucleoli with moderate amount of pale blue cytoplasm in AML M1 (Table/Fig 7)d and abnormal promyelocytes/blasts showing lobulated reniform nuclei with moderate amount of granular cytoplasm in AML M3. These cells also showed auer rods at higher magnification on BM aspiration smears in AML M3 (Table/Fig 7)e. Out of 13 cases of Acute Myeloid Leukaemia, one case was diagnosed as AML M4 on BM morphology. Few of the blasts in this case showed strong MPO positivity while others were MPO negative on a special myeloperoxidase stain. (Table/Fig 7)f. In 1 patient the BM findings were only consistent with acute leukaemia (AML-M0/ALL-L2) as only morphology and cytochemical stains were not adequate to classify it accurately and further immunophenotyping and cytogenetic studies were advised in this case. The trephine biopsy in patients with aplastic anaemia showed characteristic hypocellular BM spaces with scattered lymphocytes on haematoxylin and eosin staining (Table/Fig 7)g, while one case showed grade IV myelofibrosis on BM trephine biopsy stained with reticulin stain (Table/Fig 7)h. Six cases in this study showed increase in reticuloendothelial cell activity with increase in reticulum cells and evidences of haemophagocytosis on BM aspiration smears (Table/Fig 7)i. | |||||||||||||||||||||||||||||||||||||||||||||||||||||
DISCUSSION | |||||||||||||||||||||||||||||||||||||||||||||||||||||
Bone marrow examination is an important investigation which aids to a diagnosis and for the treatment and its prognosis. Pancytopenia is a very common haematological finding with variable clinical symptoms and signs. The various investigations like complete blood count, biochemical test, reticulocytes count, BM aspiration and biopsy, cytochemical stains, immunophenotyping, cytogenetic and molecular studies are required for arriving to a definitive diagnosis. But the key investigation remains the BM examination in patients with pancytopenia which helps in arriving to the final diagnosis as well as guiding towards further investigation and deciding the management protocols. The present study was carried out to evaluate various aetiologies of pancytopenia and to study the morphology of BM in patients with pancytopenia. Among a total of 157 patients with pancytopenia, the mean age was 40.68±23.34 years. The incidence was reported to be more in patients with age ≥18 years i.e. adult age group, and there were more males (57.32%) affected than females (42.68%). Male to female ratio was 1.34: 1.0 to a study by an almost equal gender distribution among patients with pancytopenia. Different studies from central India are shown in (Table/Fig 8) (9),(14),(15),(16),(17),(18). In the present study, the most common clinical history observed was fever followed by generalised weakness, pallor, bleeding, portal hypertension, etc. These clinical findings were found similar in Rangaswamy M et al., (17) and Varma A et al., (18). The most common sign was splenomegaly followed by hepatosplenomegaly and hepatomegaly, while there was no organomegaly in 35.7% of patients. The haemoglobin level ranged between 2.1 to 10.7 g/dL, of which most had a haemoglobin level of <7 g/dL. The total leucocyte count ranged between 100 to 3900 cells/cumm with most of the patients having the TLC between >1000 to <3500 cells/cumm and platelet count range was between 9000 to 149000 cells/cumm with maximum patients having count of <50000 cells/cumm. In terms of all the above parameters, the results of Rangaswamy M et al., (17) Kavitha M et al., (19) and Sale SM et al., (20) were comparable to the current study. The BM aspirate cellularity was classified into normocellular, hypercellular and hypocellular. Majority of the cases showed hypercellular marrow particles followed by hypocellular and normocellular in patients with pancytopenia. Similarly Gore CR et al., (21) had hypercellular BM particles as a more common finding. Rangaswamy M et al., (17) found a 75%, 14%, and 11% incidence of hypercellular, hypocellular, and normocellular BM, respectively. In patients with hypocellular and hypercellular BM particles, there was a significant difference in the p-value of TLC, while haemoglobin, platelet count and reticulocyte count did not show significant p-value. The various aetiologies in these cases were megaloblastic anaemia (n=35), hypersplenism (n=21), acute leukaemia (n=29), reactive BM with evidences of HLH (n=9), HLH (n=6), MDS (n=14), myelofibrosis (n=5), Lympho proliferative disorder (n=9) and Plasmacell dyscrasia suggestive of plasma cell myeloma (n=2). These findings also suggests that normocellular marrow fragments also harbours the characteristic morphological features pertaining to the underlying pathology and therefore all the normocellular marrow particles and cellular trails should be thoroughly examined during BM examination. The primary objective of the study was to find the causes of pancytopenia, of which the most common was megaloblastic anaemia, followed by acute leukaemia, hypersplenism, hypocellular marrow, MDS, lymphoproliferative disorder, HLH, myelofibrosis, aplastic anaemia, plasma cell dyscrasia and other causes. According to Weinzierl EP and Arber DA (22) in adults, haematologic neoplastic causes of pancytopenia were the most prevalent diagnoses, with the cases divided mostly between acute myeloid leukaemia and myelodysplastic syndrome, with fewer numbers of cases of acute lymphoblastic leukaemia, myeloproliferative neoplasms, and lymphomas. While as per Jain A et al., (23) the most common cause of pancytopenia was megaloblastic anaemia, followed by dimorphic anaemia, aplastic anaemia, and haematological malignancies. A study from Jodhpur, Rajasthan reported acute leukaemia to be the most common cause of pancytopenia, followed by megaloblastic anaemia, aplastic anaemia, hypersplenism, multiple myeloma, and myelodysplastic syndrome (24). The most common cause of pancytopenia according to Sindhiya CV and Hanagavadi S, (8) was megaloblastic anaemia, followed by combined nutritional deficiency, aplastic anaemia, iron deficiency anaemia, normal and APL. According to Chandra H et al., (25) megaloblastic anaemia, leukaemic leukemia and hypoplastic/aplastic anaemia to be the most common causes of pancytopenia. Comparison of present study with other studies with respect to aetiologies of pancytopenia has been done in (Table/Fig 9) (9),(14),(15),(16),(17),(22),(23). In this study acute lymphoid leukaemia was seen in 15 cases, out of which 1 patient had developed marrow fibrosis. There were 13 cases of acute myeloid leukaemia out of which 3 marrow aspirates showed features of myelodysplasia and probably developed from a dysplastic marrow. In the Indian subcontinent, pancytopeniais are relatively common condition that receives insufficient attention. The underlying cause of pancytopeniais are usually identified through a comprehensive clinical and haematological examination of patients with the condition. Pancytopenia, however, remains a diagnostic challenge for haematologists due to the wide range of aetiologies. Limitation(s) One of the limitation of the present study was non inclusion of dry tap/non aspirable marrow samples where the aspirationsmears were not available to study the morphology and it could be due to myelofibrosis or densely packed marrow cells. In such situations smears can be prepared from touch imprint smears of the BM trephine biopsy and the trephine biopsy which may prove helpful in arriving at the diagnosis or provides clues towards further investigations and treatment strategies. | |||||||||||||||||||||||||||||||||||||||||||||||||||||
CONCLUSION | |||||||||||||||||||||||||||||||||||||||||||||||||||||
The present study concluded that the prevlence of pancytopenia was more in males as compared to females and megaloblastic anaemia was the most common aetiology of pancytopenia The current study suggests that, for the sake of diagnosis and therapy, it is essential to recognise the factors contributing to pancytopenia and their related BM morphology, especially in emergency situations like HLH where prompt detection is important. It is imperative to rule out megaloblastic anaemia as the cause of pancytopenia because it is the leading preventable cause of patient morbidity. | |||||||||||||||||||||||||||||||||||||||||||||||||||||
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