Original article / research
Year :
2023 |
Month :
October
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Volume :
12 |
Issue :
4 |
Page :
PO66 - PO71 |
Full Version
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Diagnostic Accuracy of Peripheral Blood Film Examination in Comparison to Bone Marrow Aspiration in Various Haematological Disorders: A Cross-sectional Study
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Manjulata Gupta, Priyanka Parakh, Abhilasha Garg, Savita Soni, Anju Mishra, Dharmendra Garg 1. Assistant Professor, Department of Pathology, Rabindranath Tagore Medical College, Udaipur, Rajasthan, India.
2. Senior Demonstrator, Department of Pathology, Rabindranath Tagore Medical College, Udaipur, Rajasthan, India.
3. Assistant Professor, Department of Pathology, Rabindranath Tagore Medical College, Udaipur, Rajasthan, India.
4. Associate Professor, Department of Pathology, Government Medical College, Chittorgarh, Rajasthan, India.
5. Senior Demonstrator, Department of Pathology, Rabindranath Tagore Medical College, Udaipur, Rajasthan, India.
6. Assistant Professor, Department of Pathology, Rabindranath Tagore Medical College, Udaipur, Rajasthan, India.
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Correspondence
Address :
Manjulata Gupta, Priyanka Parakh, Abhilasha Garg, Savita Soni, Anju Mishra, Dharmendra Garg, Dr. Savita Soni,
Flat 101, A Block, Orbit Kanishka, 1/2 Bhan Bagh, New Fatehpura, Udaipur, Rajasthan, India.
E-mail: drsonisavita@gmail.com
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| ABSTRACT | | : Introduction: Bone marrow aspiration is a routine diagnostic procedure performed in hospitals for the diagnosis and management of haematological disorders. It involves cytomorphological assessment of all blood cells through bone marrow aspiration and Peripheral Blood Film (PBF) examination.
Aim: To analyse the diagnostic accuracy of PBF in comparison to bone marrow aspiration for diagnosing various haematological diseases in southern Rajasthan, India.
Materials and Methods: This cross-sectional study was conducted at the Department of Pathology, Rabindra Nath Tagore (RNT) Medical College, Udaipur Rajasthan, India, from January 2016 to June 2022. The study included microscopic findings of 638 bone marrow cases along with corresponding PBF and complete blood count findings. The results were entered into an MS Excel sheet and evaluated using Statistical Package for the Social Sciences (SPSS) 22.00 software. Chi-square test was used to calculate p-values.
Results: The study included a total of 638 bone marrow aspiration cases, with 368 cases (57.68%) being male and 270 cases (42.32%) being female. The mean age was 30.8 years (4 months to 87 years). The most common bone marrow diagnosis was Erythroid Hyperplasia, with 187 cases (29.31%) (including micronormoblastic, megalonormoblastic, normoblastic, and nutritional), followed by Chronic Myeloid Leukaemia (CML) with 154 cases (24.14%), and acute leukaemia with 152 cases (23.82%). Among the benign diagnoses, micronormoblastic erythroid hyperplasia was the most common with 68 cases (27.09%). Among the malignant diagnoses, CML was the most common with 154 cases (39.79%). The sensitivity of PBF diagnosis was 100% for Chronic Lymphocytic Leukaemia (CLL), CML, and Prolymphocytic Leukaemia (PLL), and 82.24% for acute leukaemia; 45.5% for lymphoma infiltration. The specificity was 100% for all cases. The accuracy was 95.75% for acute leukaemia, 99.06% for lymphoma infiltration, and 100% for CML, CLL, PLL, and myeloproliferative disorder-eosinophilia. The p-value was <0.001 for Acute Leukaemia, CML, CLL, PLL, myeloproliferative disorder-eosinophilia, and <0.002 for lymphoma infiltration. The overall sensitivity, specificity, accuracy, and p-value were 84.5%, 100%, 90.6%, and <0.001, respectively.
Conclusion: While bone marrow examination is crucial for confirming the diagnosis of haematological disorders, PBF examination alone can be used for phasing and confirming the diagnosis of CML and CLL. |
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Keywords
: Anaemia, Bone marrow examination, Haematological diseases, Leukaemia |
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DOI and Others
: DOI: 10.7860/NJLM/2023/63403.2806
Date of Submission: Feb 10, 2023
Date of Peer Review: May 03, 2023
Date of Acceptance: Aug 19, 2023
Date of Publishing: Oct 01, 2023
AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA
PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Feb 10, 2023
• Manual Googling: Jun 30, 2023
• iThenticate Software: Aug 18, 2023 (6%)
ETYMOLGY: Author Origin
EMENDATIONS: 8 |
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INTRODUCTION |
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Bone marrow aspiration is the most frequent and safe invasive procedure routinely performed in hospitals to evaluate various neoplastic and non neoplastic haematological disorders (1). It is done after a proper assessment of medical history, physical examination, blood count analysis, and examination of peripheral smear morphology (2). It is very helpful in confirming clinically suspected diseases and providing previously unsuspected diagnoses (3).
The concomitant examination of bone marrow aspiration and PBF examination is conclusive as it provides information regarding the morphology and maturation of all lineages of blood cells (4). It also helps in finding out prognosis based on staging and assessing disease staging before and after chemotherapy (5). Other analyses such as flow cytometry, microbiological tests, cytogenetics, molecular genetics, and immunophenotyping can also be performed on bone marrow aspirate (2),(6). In various haematological diseases, there is a disordered numerical and spatial relationship among bone marrow cells and changes in cell appearance. The spectrum of haematological disorders diagnosed with bone marrow aspiration examination ranges from common disorders like nutritional deficiency anaemia to life-threatening conditions like leukaemia (4).
Bone marrow aspiration can be performed in severe thrombocytopenia with a minimum risk of bleeding (1). However, many contraindications also exist for this procedure such as Haemorrhagic disorders, disseminated intravascular coagulation, anticoagulant drugs; sampling site skin infection, and bone disorders such as osteomyelitis or osteogenesis imperfecta (7). This study analysed the diagnostic accuracy of PBF in comparison to bone marrow aspiration for diagnosing various haematological diseases and to study the spectrum of various haematological diseases in different age groups in southern Rajasthan, India. The study helps to establish the use of PBF for diagnosing various haematological malignancies in resource-poor settings.
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Material and Methods |
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The present study was a cross-sectional study conducted from January 2016 to June 2022 in Department of Pathology, Rabindranath Tagore Medical College and Associated Hospitals in Udaipur, Rajasthan, India. A total of 638 bone marrow aspiration cases received in the haematology laboratory during that duration were included in the study. As it was a laboratory-based cross-sectional study, there was less than minimal risk to the patients, and the data related to identity were kept highly confidential. Clinical data, including the mode of onset, history of previous illness and treatment, bone pains, fever, bleeding, hepatosplenomegaly, lymphadenopathy, Complete Blood Cell (CBC) count, and PBF, were recorded.
Inclusion criteria: All bone marrow aspiration smears with available CBC count and PBF were included in the study.
Exclusion criteria: All unsatisfactory diluted bone marrow aspiration smears and smears with non availability of clinical history and relevant data were excluded from the study.
Preparation of a peripheral blood smear: About 2 to 3 mL of blood was collected in an Ethylenediaminetetraacetic Acid (EDTA) anticoagulated vial from the antecubital vein. A drop of blood was taken and placed on a clean glass slide, 1 cm from the end. A smear was made using the wedge method, creating tongue-shaped smears of about 2.5 to 3.5 cm in length. At least two smears were prepared. The smears were air-dried, fixed in methanol, and stained with an in-house made Giemsa stain. Bone marrow aspiration was done using Salah’s bone marrow puncture needle. The preferred site was the posterior superior iliac spine. Smears were made, air-dried, fixed in methanol, and stained with Giemsa stain. PBF findings were compared with bone marrow aspiration results, as bone marrow biopsy was available for very few cases.
Microscopic examination of smears: PBF examination included a low-power scan to check for the presence of hemoparasites, a 40x examination to assess Red Blood Cell (RBC) and White Blood Cell (WBC) morphology and count, and an oil immersion examination to evaluate blast cell morphology and platelet count. Bone marrow smear examination involved a low-power scan to assess sample adequacy, as well as high power and oil immersion lens examinations to determine cellular morphology and distribution. The smears were assessed for cellularity, differentiation, and maturation of the erythroid, myeloid, and megakaryocytic lineages, as well as the presence of plasma cells, lymphocytes, parasites/abnormal cells/granulomas/storage cells. The myeloid: erythroid ratio was calculated, and malignant haematological disorders were categorised using the 5th edition of the WHO classification of haematolymphoid tumours [8,9].
Statistical Analysis
The collected data was entered into an Excel sheet and analysed using SPSS version 22.00 software. The mean, specificity, sensitivity, p-value using the chi-square test, and accuracy were calculated.
Senstivity=(True Positive/True Positive +False Negative) *100
Specificity=(True Negative/False Positive+True Negative) *100
Accuracy=(True positive+True Negative/True Positive+True Negative+
False Positive+False Negative) *100
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Results |
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Out of the 638 cases included in the study, 368 (57.68%) were male and 270 (42.32%) were female (Table/Fig 1). The mean age was 30.8 years (4 months to 87 years).
As depicted in (Table/Fig 2),(Table/Fig 3), hypoplastic anaemia (18 cases), anaemia’s including megaloblastic (46 cases), micronormoblastic (37 cases), and nutritional anaemias (33 cases), as well as ITP (11 cases) with acute leukaemia (123 cases), showed the highest number of cases in the 0-20 years age group. The number of cases of CML was 78, and plasma cell dyscrasias had 11 cases, with the highest occurrence observed in the 41-60 years age group. CLL was more common in the 61-80 years age group, with 26 cases reported.
The highest number of diagnoses based on PBF results were CML with 154 cases (24.1%), followed by pancytopenia with 147 cases (23.04%), acute leukaemia with 125 cases (19.6%), and exclusive anaemia with 94 cases (14.73%), including dimorphic, microcytic, macrocytic, and normocytic anaemias (Table/Fig 1).
Regarding bone marrow diagnoses, the highest number of cases were erythroid hyperplasia with 187 cases (29.31%), including micronormoblastic, megalonormoblastic, normoblastic, and nutritional types. This was followed by CML with 154 cases (24.14%), and acute leukaemia with 152 cases (23.82%), as presented in (Table/Fig 2),(Table/Fig 3).
Among the benign bone marrow diagnoses, the highest number of cases were micronormoblastic erythroid hyperplasia with 68 cases (27.09%), followed by megalonormoblastic erythroid hyperplasia with 59 cases (23.51%), and nutritional anaemia with 44 cases (17.53%), as shown in (Table/Fig 4).
In malignant bone marrow diagnoses, the highest number of cases were CML with 154 cases (39.79%), followed by acute leukaemia with 152 cases (39.27%), CLL with 41 cases (10.6%), and plasma cell dyscrasia with 16 cases (4.14%), as shown in (Table/Fig 5),(Table/Fig 6),(Table/Fig 7). The sensitivity, specificity, accuracy, and p-value of PBF examination results for certain malignant haematological disorders were calculated, considering bone marrow aspiration smear examination as the gold standard.
The sensitivity of PBF examination was 100% for CLL, CML, and PLL; 82.24% for acute leukaemia; 45.5% for lymphoma infiltration, and 0% for Immune Thrombocytopenic Purpura (ITP), plasma cell dyscrasia, and metastasis. The specificity was 100% for all cases, as shown in (Table/Fig 8).
The accuracy was 95.75% for acute leukaemia, 99.06% for lymphoma infiltration, and 100% for CML, CLL, PLL, and myeloproliferative disorder-eosinophilia. The p-value was <0.001 for acute leukaemia, CML, CLL, PLL, and myeloproliferative disorder-eosinophilia, and <0.002 for lymphoma infiltration, indicating statistical significance. The overall sensitivity, specificity, accuracy, and p-value of PBF for diagnosing malignant cases were 84.5%, 100%, 90.6%, and <0.001, respectively.
Using Giemsa stain, the cytomorphological features of ITP and plasma cell dyscrasia were studied, as shown in (Table/Fig 9),(Table/Fig 10).
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Discussion |
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In this study, there were 251 cases (39.34%) of benign haematological diagnoses, while 387 cases (60.66%) were malignant diagnoses. The number of malignant cases was higher compared to other similar studies such as Gandapur AS et al., (27.8%) and Bagale P and Shingade M (22%) because bone marrow aspiration was preferentially performed on suspicious malignant cases (10),(11).
This study showed a male preponderance with a male-to-female ratio of 4:3, which was comparable to studies conducted by Raina JS et al., (3:2), Manzoor F et al., (1.6:1), and Biradar MP et al., (2:1) (6),(12),(13). Male preponderance was in accordance with clinical-epidemiological studies, which have stated that men are more prone to haematological disorders.
According to this study, the age of cases ranged from 4 months to 87 years, which was comparable to the studies conducted by Raina JS et al., (1 year to 90 years), Das S et al., (4 years to 87 years), Gayathri BN and Rao KS (2 to 80 years) (2011), and Vaidya S (4 to 75 years) (2015) (6),(14),(15),(16). The age group with the highest number undergoing bone marrow aspiration was 0-20 years (297 cases, 46.55%), whereas Shastri SM and Kolte SS had 28 cases (25.45%) in the 21-30 years age group and 22 cases (20%) in the 11-20 years age group (17). As depicted in (Table/Fig 2), cases of hypoplastic anaemia, anaemias including megaloblastic, micronormoblastic, and nutritional anaemias, and ITP with acute leukaemia showed the highest number of cases in the 0-20 years age group.
CML and plasma cell dyscrasias were most common in the 41-60 years age group, whereas CLL was common in the 60-80 years age group. Erythroid hyperplasia was the most common diagnosis in bone marrow aspiration, accounting for a total of 29.31% in present study. Similar results were seen in studies by Pudasaini S et al., (21%), Khodke K et al., (14%), and Jha A et al., (19.6%) (1),(18),(19).
Micronormoblastic erythroid hyperplasia was observed in 68 cases (10.66%) on bone marrow aspiration, compared to a study by Al-Ghazaly J et al., (10.4%). It had the highest incidence in the 0-20 years age group and was associated with malnutrition, parasitic infestation, gastrointestinal haemorrhage, and heavy menstrual bleeding (20). Microcytic anaemia and pancytopenia were the most common concomitant findings on PBF examination.
Megalonormoblastic erythroid hyperplasia was seen in 59 cases (9.25%), compared to other studies done by Pudasaini S et al., and Khodke K et al., Al-Ghazaly J et al., Ahmed SQ et al., which showed (12.3%), (6.5%), (9.1%), and (11.9%) of their cases, respectively (1),(18),(20),(21).
Pancytopenia was the most common concomitant finding on PBF examination. Megaloblastic anaemia may be associated with a vegetarian diet, chronic diarrhoea, malabsorption, and malnutrition due to a low socio-economic status. It can also be caused by hereditary haemolytic anaemias in aplastic crises, pernicious anaemia, and tropical splenomegaly syndrome caused by recurrent malaria infestation leading to folic acid deficiency. Most patients presented with pancytopenia, which necessitated bone marrow aspiration for diagnosis. Concomitant vitamin B12 and folic acid assays were not performed.
Nutritional deficiency, comprising megalonormoblastic and micronormoblastic erythroid hyperplasia, was seen in 44 cases (6.90%), similar to the study by Balasubramanian M and Sangoi NN (13.3%) (4). Pancytopenia was the most common finding associated with this condition on PBF examination. It could be attributed to low socioe-conomic status and a vegetarian diet. Hypoplastic anaemia was seen in 18 cases (2.82%), comparable to studies by Pudasaini S et al., (5.3%), Gandapur AS et al., (1.75%), and Bagale P and Shingade M (5.92%) (1),(10),(11). In all cases of hypoplastic anaemia, the marrow was hypocellular and all three lineages of cells were suppressed. BMA findings were correlated with PBF, which also showed pancytopenia in these cases (Table/Fig 5). However, bone marrow biopsy was not performed in these cases.
In this study, acute leukaemia comprised 152 cases (23.82%), similar to the study by Balasubramanian M and Sangoi NN (27.3%) (4). Various factors such as differences in methodology, geographic distribution, genetic disturbances, preferential aspiration of suspicious leukaemia cases, and the period of observation may cause variation in the incidence of disorders causing acute leukaemia. The predominant reason for not giving a conclusive diagnosis of acute leukaemia on PBF examination in present cases was pancytopenia, bicytopenia, and subleukaemic leukaemia in decreasing order. Both PBF and bone marrow aspiration were complementary in all other cases of leukaemia diagnosed in this study.
According to this study, CML comprised the highest number of malignant diagnoses with 154 cases (24.14%), which was higher compared to other similar studies. This could be attributed to differences in patients’ lifestyle and economic status, as well as the preferential availability of treatment for CML in our center compared to other leukaemias. The high frequency of leukaemia, especially in rural areas and less in urban areas, may be due to the use of pesticides in agriculture without regulations and protective measures.
In the present study, CLL comprised 41 cases (6.42%), which was similar to the study by Al-Ghazaly J et al., (5.7%) and Nasher ST et al., (3.7%) (22),(20). Plasma cell dyscrasia comprised 16 cases (2.51%), similar to the study by Pudasaini S et al., (3.5%) (1) and Jha A et al., (0.94% to 4.1%) (19), and Bagale P and Shingade M (2.22%) (11). Both CLL and plasma cell dyscrasia were mostly seen in the older age group. Most CLL cases were diagnosed incidentally, while plasma cell dyscrasia cases presented with pathological fractures. CLL and plasma cell dyscrasia were more commonly seen in males. Myelodysplastic Syndrome (MDS) comprised four cases (0.63%), similar to Bagale P and Shingade M (0.6%) (11) and Pudasaini S et al., (3.5%) (1). The diagnosis of MDS was given after excluding all other secondary causes of dysplasia. ITP was diagnosed in 14 cases (2.19%), similar to the study by Khodke K et al., (5%) (18). The present study showed the maximum number of ITP cases in the 0-20 years age group, mostly in children, associated with viral infections and autoimmunity.
Infective pathology was seen in six cases, out of which five cases were diagnosed with Malarial infection and one case was diagnosed with Leishmaniasis. This study showed 0.78% of malaria cases, similar to Santra G and Das BK (1.8%) (23). All the cases were documented during the summer to rainy season, corresponding to the prominent mosquito breeding season. Only one case (0.16%) of Leishmaniasis was seen, similar to Bagale P and Shingade M (0.25%) (11). The patient was a migrant laborer from Nepal. Granuloma was seen in two cases (0.31% cases), similar to Das S et al., (1.16%) (14). Both patients had a history of tuberculosis.
Lymphoma infiltration was seen in 11 cases (1.72%), similar to Dogan A et al., (2%) (2). Peripheral blood involvement was seen in five cases (45.4%) out of the 11 cases with marrow involvement, similar to the studies by Goel N et al., and Sovani V et al., (7),(24). On follow-up, all the cases were found to be non Hodgkin’s lymphoma. Metastasis was seen in seven cases (1.09%), similar to Dogan A et al., (1.8%) (2). There were 17 cases showing normal marrow findings, which were mostly associated with normal PBF findings (six cases). These cases were suspected cases of multiple myeloma and metastasis. Other associated cases were unexplained normocytic normochromic anaemia (five cases), eosinophilia (two cases), microcytic hypochromic anaemia (two cases), and one case each of neutrophilia and pancytopenia.
The diseases diagnosed on bone marrow aspiration in cases where PBF was inconclusive were metastatic cancers, hypoplastic anaemias, lymphoma infiltration, plasma cell dyscrasia, ITP, and granuloma. Hence, for these diagnoses, bone marrow aspiration is essential (Table/Fig 5),(Table/Fig 6). PBF examination was conclusive and showed 100% sensitivity for the diagnosis of CLL, PLL, and CML, whereas the specificity of PBF examination was 100% for all diagnoses (Table/Fig 4). The overall sensitivity of 84.5% and accuracy of 90.6% were due to the inability of PBF examination to diagnose all cases of plasma cell dyscrasia, metastasis, MDS, and a few cases of lymphoma infiltration and leukaemias. The sensitivity of 100% ensures that PBF can exclude all cases not having haematological malignancy. The p-value of <0.001 is statistically significant. The calculated accuracy, sensitivity, specificity, and p-value show that PBF examination is a useful procedure to detect CML, CLL, PLL, and myeloproliferative disorder, but it cannot be a substitute for examination of the marrow by bone marrow aspiration and core biopsy in all other cases. The utility of PBF examination is supported by its earlier and easier availability, although the combination of PBF and bone marrow examination is best to detect all the true positives and limit false-negative diagnoses.
Limitation(s)
Bone marrow biopsy was done for only a few cases as bone marrow aspiration provides a sample for both cytomorphology and ancillary testing (such as flow cytometry) and gives an early diagnosis within days compared to biopsy, whose results take weeks to assess.
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Conclusion |
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Marrow aspirate is a simple, rapid method primarily utilised for cytological assessment. It is a decisive step for the confirmatory diagnosis of haematological disorders. Bone marrow examination gives confirmatory pathological diagnosis for unexplained cytopenias and leukaemia. It also gives the type of erythroid reaction to anaemia that cannot be determined from the peripheral blood smear alone. From present study, it can be concluded that for the phasing and confirmatory diagnosis of CML and CLL, PBF examination alone was found to be sufficient.
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TABLES AND FIGURES | |
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