Bone Marrow Aspiration Smears Research Articles

Population structures of Leishmania infantum and Leishmania tropica the causative agents of kala-azar in Southwest Iran

Visceral leishmaniasis (VL) is endemic in Iran and is caused predominantly by Leishmania infantum, but L. tropica is emerging as an important cause. We studied the intra-species population structure of Leishmania spp. causing VL in southwest Iran by sequence analysis of the internal transcribed spacer (ITS) 1 of DNA samples from 29 bone marrow aspiration smears. L. infantum (n = 25) and L. tropica (n = 4) were identified, consisting of 10 and three ITS1 sequence types (STs), respectively. Compared to GenBank ITS1 STs, our L. infantum parasites displayed high heterogeneity but less heterogeneity compared than northwest Iranian isolates. VL affects mostly nomadic populations in southwest Iran, and their mobility may explain partly the L. infantum heterogeneity. The VL causing L. tropica was also genetically heterogeneous but genetically indistinguishable from L. tropica strains causing anthroponotic cutaneous leishmaniasis from southwest Iran.

Evaluation of pancytopenia on bone marrow aspiration- study at a tertiary care center in Kashmir valley, India

Background: Pancytopenia refers to combination of anaemia, leukopenia and thrombocytopenia. It may be a manifestation of a wide variety of disorders, which primarily or secondarily affect the bone marrow. However, aetiology of pancytopenia varies from one geographical region to another. Bone marrow aspiration plays an important role in identifying the cause of pancytopenia. This study was carried to identify the causes of pancytopenia and to find out the bone marrow morphology in cases of pancytopenia.Methods: This study was conducted in the department of haematology in a tertiary care center in Kashmir valley for a period of 3 years. Inclusion criteria: cases with hemoglobin less than 10 gm/dl, total leucocyte count of less than 4000/mm3 and platelet count less than 100,000/mm3 were included in the study.Exclusion criteria: Patients receiving chemotherapy/radiotherapy were excluded from the study. Bone marrow aspiration (BMA) was performed from posterior iliac crest of the patients. Bone marrow aspiration smears were stained with Leishman stain for microscopy.Results: A total of 334 cases were studied during a period of 3 years. Age of patients ranged from 1 year to 85 years with mean of 43.59 years.180 cases were male, and 154 cases were female with male:female ratio of 1.2 :1. The commonest cause of pancytopenia was megaloblastic anemia seen in 103 cases (30.8%) followed by dual deficiency anemia seen in 69 cases (20.7%).37 cases (11%) were of acute leukaemia. Aplastic anemia was seen in 35 cases(10.5%). Other causes of pancytopenia were myelodysplastic syndrome, multiple myeloma, iron deficiency anemia and hypersplenism.Conclusions: Bone marrow aspiration in patients of pancytopenia helps in the identification of the underlying cause in most of the cases. BMA is helpful for understanding the disease process; and in planning further investigations and management of cytopenia patients.

Super-Resolution Digital Pathology Image Processing of Bone Marrow Aspirate and Cytology Smears and Tissue Sections

Background: Accurate digital pathology image analysis depends on high-quality images. As such, it is imperative to obtain digital images with high resolution for downstream data analysis. While hematoxylin and eosin (H&E)-stained tissue section slides from solid tumors contain three-dimensional information, these data have been ignored in digital pathology. In addition, in cytology and bone marrow aspirate smears, the three-dimensional nature of the specimen has precluded efficient analysis of such morphologic data. An individual image snapshot at a single focal distance is often not sufficient for accurate diagnoses and multiple whole-slide images at different focal distances are necessary for diagnostics. Materials and Methods: We describe a novel computational pipeline and processing program for obtaining a super-resolved image from multiple static images at different z-planes in overlapping but separate frames. This program, MULTI-Z, performs image alignment, Gaussian smoothing, and Laplacian filtering to construct a final super-resolution image from multiple images. Results: We applied this algorithm and program to images of cytology and H&E-stained sections and demonstrated significant improvements in both resolution and image quality by objective data analyses (24% increase in sharpness and focus). Conclusions: With the use of our program, super-resolved images of cytology and H&E-stained tissue sections can be obtained to potentially allow for more optimal downstream computational analysis. This method is applicable to whole-slide scanned images.

Beware of Bone Marrow: Incidental Detection and Primary Diagnosis of Solid Tumours in Bone Marrow Aspiration and Biopsies; A Study of 22 Cases

Introduction: First detection of any solid tumour as metastatic deposits in bone marrow directs clinicians to start searching for the primary tumour. Detection of bone marrow metastasis determines the stage of the malignancy, prognosis, mode of treatment, chemotherapeutic response and follow-up in case of relapse. The aim of the current study was to analyse the clinico-haematological presentation and morphological pattern of infiltration of solid tumours detected first as metastatic deposits on bone marrow examination. Three-year retrospective analysis of MGG-stained bone marrow aspiration smears and touch imprints of the bone marrow biopsy and Hematoxylin and Eosin (H&E) stained histopathological sections of biopsies was performed at the Department of Pathology at a tertiary care institute (January 2014 to December 2016). The morphological pattern of metastatic deposits and haematological profiles of the patients were analysed. Exclusion criterion was the presence of hematolymphoid malignancies. In 22 cases, bone marrow was the first site of detection of metastasis. The age of the patients ranged from 3 years and 10 months to 82 years, with equal gender predilection. Overall, 16 cases had cytopenias, 9 cases each had leucocytosis and leukoerythroblastic presentation. The metastasis was from Ewing's sarcoma, prostate carcinoma, gastric adenocarcinoma invasive duct carcinoma breast, gallbladder carcinoma, lacrimal duct carcinoma and invasive papillary urothelial carcinoma. Bone marrow examination is a cheap and reliable investigation to detect metastasis in an unsuspecting case. Bilateral trephine biopsies are recommended to increase the efficacy of detecting bone marrow metastasis.

Lysosomal storage disorders: Morphologic appraisal in Indian population.

Lysosomal storage disorders (LSDs) comprise a group of at least 50 distinct genetic diseases, each one resulting from the deficiency of a particular lysosomal enzyme involved in metabolism. We attempt to study and further subclassify pediatric LSDs into Gaucher's and non-Gaucher's category based on the morphologic variables seen in the bone marrow aspiration smears and trephine biopsy sections. Pediatric (<12 years age) cases of LSDs diagnosed by bone marrow aspiration and trephine biopsy specimens, in the last 12 years period, were retrieved. The archival material and the relevant clinical as well as hematologic parameters were reviewed. From January 1997 to December 2008, 55 cases were diagnosed as LSDs. Based on bone marrow morphology, 56% (n = 31) cases were diagnosed as non-Gaucher's and the remaining 44% (n = 24) cases as Gaucher's disease, the ratio being 1.29:1. Anemia and thrombocytopenia were more commonly observed in Gaucher's disease (91.67 and 62.5%) as compared to non-Gaucher's group (74.19 and 19.35%). Neurologic symptoms and signs were more frequently present in non-Gaucher's cases (45.16%) as compared to Gaucher's group (29.17%). LSDs can be classified into Gaucher's and non-Gaucher's subtypes based on the characteristic cytomorphology of the storage cells in Giemsa-stained bone marrow aspiration smears and on hematoxylin and eosin-stained trephine biopsy sections. This approach would be fairly adequate for therapeutic and prognostic purposes in resource.constrained settings, where enzyme studies and mutational analysis may not be easily available.

A study of megakaryocyte morphology in bone marrow aspiration smears of cases of thrombocytopenia

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.

Comparison of Clinical Outcomes Between Pediatric Aplastic Anemia and Refractory Cytopenia of Childhood

In the diagnosis of childhood bone marrow failures (BMFs), differentiating aplastic anemia (AA) from hypoplastic myelodysplastic syndrome (MDS) is challenging. The 2008 World Health Organization (WHO) classification has proposed a provisional entity, "refractory cytopenia of childhood (RCC)". The spectrum of patients with RCC is wide, ranging from patients with severe hypocellular bone marrow (BM) and mild dysplasia to those with normocellular BM and distinct dysplasia meeting the criteria for refractory cytopenia with multilineage dysplasia (RCMD) defined for adults with MDS. Currently, it is recommended that children who meet the criteria for RCMD should be classified as RCC in the WHO classification until the number of lineages involved has been fully evaluated with regard to their relative importance as prognostic factors. Until now, few studies have addressed the question whether the current WHO classification reflects clinical outcomes of childhood BMFs.To determine the clinical differences among AA, RCC, and RCMD, we compared clinical outcomes for patients with AA, RCC, and RCMD in Japan. From February 2009 to December 2013, 252 patients were registered to the central morphology review system of the Japanese Society of Hematology and Oncology and were diagnosed with BMFs. Peripheral blood (PB) and BM smears were reviewed by two pediatric hematologists, and BM trephine biopsies were reviewed by a hematopathologist. RCC is defined as persistent cytopenia with <2% and <5% blasts in PB and BM, respectively. BM aspirate smears show dysplastic changes in >2 cell lineages or >10% within one cell lineage. On the other hand, the criteria of RCMD is defined as persistent cytopenia with <1% and <5% blasts in PB and BM, respectively. BM smears show >10% dysplastic changes in >2 cell lineages. Patients with inherited BMFs were excluded by family history and physical examination. Further, Fanconi anemia was excluded by chromosome fragility test and Dyskeratosis congenita was screened by measuring the telomere length of the peripheral lymphocytes by flowcytometry.Out of 252 patients, 63 were classified as AA, 131 as RCC, and 58 as RCMD. Median ages in AA, RCC, and RCMD groups were 10, 8, and 7 years, respectively (p=0.07). The median of leukocyte, neutrophil, reticulocyte, and platelet count, and mean corpuscular volume were significantly lower in AA than in RCC and RCMD groups (p<0.01). Chromosomal abnormalities were detected in 1 patient with AA (trisomy 8), 3 patients with RCC (trisomy 8, n=2; other, n=1), and 9 patients with RCMD (trisomy 8, n=5; monosomy 7, n=1; other, n=3) at the time of diagnosis (p<0.01). Out of 252 patients, 82 (AA, n=3; RCC, n=46; RCMD, n=33) were observed without any treatments (watch and wait, WW). 5-year overall survival (OS)/failure free survival (FFS) rates in WW group were 67%/67% in AA, 98%/54% in RCC, and 100%/69% in RCMD patients (p<0.01/p=0.97). Immunosuppressive therapy (IST) with rabbit antithymocyte globulin and cyclosporine was performed in 110 (AA, n=39; RCC, n=57; RCMD, n=14) patients. Six months after the IST, the response rates to the IST were not significantly different among AA (40%), RCC (63%), and RCMD (64%) (p=0.08). The development of additional chromosomal aberrations was found in 2 patients with RCC, and 1 with RCMD. The 5-year OS/FFS rates in IST group were 89%/36% in AA, 94%/38% in RCC, and 93%/23% in RCMD patients (p=0.64/p=0.86). Stem cell transplantation (SCT) as a first line therapy was performed in 19 patients with AA, 10 with RCC, and 5 with RCMD. The rejection was found in 2 patients with RCC and 3 with RCMD. Although 5-year OS rates in patients who underwent SCT were not different among 3 groups (p=0.26), FFS rate (30%) in patients with RCMD was significantly lower than in those with AA (100%) and RCC (78%) (p<0.01).In conclusion, we could not find any clinical relevance of separating RCC from AA because response rates to IST and the development of clonal evolution did not significantly differ between AA and RCC. The entity of RCMD should be adopted to childhood MDS classification because children with RCMD exhibited a distinct characteristic of morphology and a frequent chromosomal aberration at the time of diagnosis. The optimal treatment strategy including preconditioning regimen of SCT should be established for children with acquired BMFs based on the BM cellularity and morphological classification. DisclosuresKojima:SANOFI: Honoraria, Research Funding.

Bone marrow aspiration study of megakaryocytic alterations in non myelodysplastic syndrome related thrombocytopenia

Background: Thrombocytopenia is a common hematological condition associated with variable etiological factors. Dysplastic changes of megakaryocytes in thrombocytopenia are commonly seen in myelodysplastic syndrome (MDS). However, several studies have described it’s occurrence in non-myelodysplastic hematological conditions. The present study was undertaken to note the various morphological alterations in megakaryocytes (including both dysplastic and non dysplastic changes) in non MDS related thrombocytopenia. Materials and Methods: A prospective study of 78 cases with thrombocytopenia were included in the present study. Cases of MDS were excluded from the study. Informed consent was taken from all the patients with thrombocytopenia and Bone marrow aspiration was done in all cases. Bone marrow aspiration smears were air dried and stained with Leishman stain. Bone marrow aspiration smears were examined for number and various morphological alterations. Special investigations were done in required cases for confirmation of diagnosis. Results: Dysplastic megakaryocytes were observed in 78.6% cases of immune thrombocytopenic purpura and 41.2% cases of megaloblastic anemia. Most common dysplastic feature observed was micromegakaryocyte (32.0%) followed by multiple separate nuclei (21.7%) and hypogranular form was the least observed dysplastic feature (8.9%). Conclusions: Dysplasia in megakaryocytes is a common finding in various non-myelodysplastic syndrome related thrombocytopenia. The mere presence of dysplastic megakaryocyte should not prompt an interpretation of myelodysplastic syndrome and should always be correlated with patient’s clinical and other hematological parameters.

Automatic recognition of myeloma cells in microscopic images using bottleneck algorithm, modified watershed and SVM classifier.

Plasma cells are developed from B lymphocytes, a type of white blood cells that is generated in the bone marrow. The plasma cells produce antibodies to fight with bacteria and viruses and stop infection and disease. Multiple myeloma is a cancer of plasma cells that collections of abnormal plasma cells (myeloma cells) accumulate in the bone marrow. The definitive diagnosis of multiple myeloma is done by searching for myeloma cells in the bone marrow slides through a microscope. Diagnosis of myeloma cells from bone marrow smears is a subjective and time-consuming task for pathologists. Also, because of depending on final decision on human eye and opinion, error risk in decision may occur. Sometimes, existence of infection in body causes plasma cell's increment which could be diagnosed wrongly as multiple myeloma. The computer diagnostic process will reduce the diagnostic time and also can be worked as a second opinion for pathologists. This study presents a computer-aided diagnostic method for myeloma cells diagnosis from bone marrow smears. At first, white blood cells consist of plasma cells and other marrow cells are separated from the red blood cells and background. Then, plasma cells are detected from other marrow cells by feature extraction and series of decision rules. Finally, normal plasma cells and myeloma cells could be classified easily by a classifier. This algorithm is applied on 50 digital images that are provided from bone marrow aspiration smears. These images contain 678 cells: 132 normal plasma cells, 256 myeloma cells and 290 other types of marrow cells. Applying the computer-aided diagnostic method for identifying myeloma cells on provided database showed a sensitivity of 96.52%; specificity of 93.04% and precision of 95.28%.

Comparison of Clinical and Laboratory Features Between Pediatric Aplastic Anemia and Refractory Cytopenia of Childhood

In the diagnosis of childhood bone marrow failure (BMF), differentiating aplastic anemia (AA) from hypoplastic myelodysplastic syndrome (MDS) is challenging. The 2008 World Health Organization (WHO) classification has proposed a provisional entity, "refractory cytopenia of childhood (RCC)". The spectrum of patients with RCC is wide, ranging from patients with severe hypocellular bone marrow (BM) and mild dysplasia to those with normocellular BM and distinct dysplasia meeting the criteria of refractory cytopenia with multilineage dysplasia (RCMD) defined for adults with MDS. Currently, it is recommended that children who meet the criteria for RCMD should be classified as RCC in the WHO classification until the number of lineages involved has been fully evaluated with regard to their relative importance as prognostic factors. Until now, no studies have addressed the question whether the current WHO classification reflects clinical outcomes of childhood BMF.The Japanese Society of Pediatric Hematology and Oncology has established a central review system of morphology. Peripheral blood (PB) and BM smears were reviewed by two pediatric hematologists, and BM trephine biopsies were reviewed by a hematopathologist. In addition, the telomere length of lymphocytes and paroxysmal nocturnal hemoglobinuria (PNH) clones in PB were measured by flowcytometry. RCC is defined as persistent cytopenia with <2% and <5% blasts in PB and BM, respectively. BM aspirate smears show dysplastic changes in >2 cell lineages or >10% within one cell lineage. On the other hand, the criteria of RCMD is defined as persistent cytopenia with <1% and <5% blasts in PB and BM, respectively. BM smears show >10% dysplastic changes in >2 cell lineages. We classified childhood BMF into AA, RCC, and RCMD in our central review.From February 2009 to February 2015, 1,300 cases were prospectively reviewed. Of the 1,300 cases, 582 were classified as BMF. Among them, 99 were classified as AA, 230 as RCC, 128 as RCMD, 50 as hepatitis-related BMF, 4 as PNH, and 71 as inherited BMF. Of the 71 cases with inherited BMF, 35 were diagnosed as Fanconi anemia, 14 as Shwachman-Diamond syndrome, and 12 as dyskeratosis congenita. To determine the clinical differences among AA, RCC, and RCMD, we compared laboratory and clinical findings for 457 patients classified with AA, RCC, and RCMD. Median ages in the AA, RCC, and RCMD groups were 10, 9, and 7 years, respectively (p<0.01). The male/female ratio in AA, RCC, and RCMD groups was 1.3, 1.4, and 1.1. The median of leukocyte, neutrophil, reticulocyte, and platelet count, and mean corpuscular volume were significantly lower in AA than in RCC and RCMD groups (p<0.01). Chromosomal abnormalities were detected in 1 patient with AA (trisomy 8), 6 patients with RCC (trisomy 8, n=4; monosomy 7, n=1; other, n=1), and 18 patients with RCMD (trisomy 8, n=5; monosomy 7, n=4; other, n=9) (p<0.01). Out of the 457 patients, 69 (AA, n=29; RCC, n=34; RCMD, n=6) were treated with immunosuppressive therapy (IST) with rabbit antithymocyte globulin and cyclosporine. Six months after the IST, the response rate to the IST was not significantly different between AA (38%) and RCC (47%) (p=0.47). Although the number of patients was limited, the response rate to the IST was 100% in patients with RCMD. One patient with RCC developed PNH at 26 months after the IST. Stem cell transplantation was performed in 15 patients with AA, 16 with RCC, and 3 with RCMD. Two patients with AA and 1 patient with RCC died of transplant-related toxicities.In conclusion, the entity of RCMD should be adopted to childhood MDS classification because children with RCMD exhibited a distinct characteristic of morphology and laboratory findings. Further efforts to clarify molecular events and clinical outcomes are necessary to establish the classification system for childhood BMF. DisclosuresNo relevant conflicts of interest to declare.

Bone marrow aspirate microscopy v. bone marrow trephine biopsy microscopy for detection of Mycobacterium tuberculosis infection.

Tuberculosis (TB) remains a global health problem. According to the 2013 Global Report on Tuberculosis, 8.6 million people developed TB in 2012 and 1.3 million died from the disease. An estimated 13% of people who developed TB in 2012 were HIV-positive, and 75% of these lived in Africa. While pulmonary TB is the commonest form of Mycobacterium tuberculosis infection, extrapulmonary TB is increasingly being detected in HIV-positive patients. Definitive diagnosis of disseminated TB is a challenge owing to atypical presentations and diagnostic difficulties (negative chest radiograph and sputum microscopy and culture). A rapid diagnosis of disseminated TB is desirable, as early initiation of treatment can reduce mortality. Although TB culture is the gold standard for diagnosis of TB, it has a long turnaround time (up to 6 weeks). To identify a potentially faster and more effective diagnostic strategy for disseminated TB. A retrospective 18-month review, conducted at a tertiary hospital, comparing histological findings of an auramine O-stained bone marrow aspiration (BMA) smear and a bone marrow trephine (BMT) biopsy specimen with the gold standard of TB culture. Microscopic examination of BMA smears and BMT biopsy specimens offers a rapid diagnostic strategy, with results available on the same day for the former and within 4 days for the latter. BMT histological examination had a significantly higher detection rate than BMA auramine O staining compared with TB culture. We recommend that BMT biopsies remain an essential part of the diagnostic work-up for disseminated TB.

Bone marrow touch imprint smears as an adjunct to bone marrow aspiration smears in hematological disorders

Background: Morphological examination of the marrow requires a combination of a properly prepared bone marrow aspirate smear, a trephine biopsy section and an imprint of core biopsy. Some conditions often result in a dry tap and are best studied by marrow biopsy.The major drawbacks of biopsy sections are their thickness, precludingfine morphologic detail.The objective of this study was to compare the diagnostic accuracy, cellularity and cytomorphology from bone marrow biopsy core imprint smears with bone marrow aspiration smears.Materials and Methods: Imprint smears were prepared from 138 cases subjected to bone marrow examination. The bone marrow aspiration, imprint smears andbone marrow biopsy sections were examined and were categorized into five different groups on cytomorphological basis: Non-malignant alterations and normal marrow, Myeloid neoplasms, Plasma cell myeloma, myelo-infiltrative disease and absence of residual disease and further delineated into specific entities wherever necessary.Results: Out of 138 cases, non-malignant alterations and normal marrow was the largest subgroup (N=87, 63%), followed by myeloid neoplasms (N=26, 18.5%), Plasma cell myeloma (N=13, 9.4%), myelo-infiltrative disease (N=9, 6.5%) and absence of residual disease (N=3, 2.2%). The diagnostic accuracy of imprint smears was highest (92%) followed by biopsy sections (89.9%) and aspiration smears (87%). Kappa analysis showed strong agreement (&gt;0.8) and p-value was statistically significant (&lt;0.001) while correlating the final diagnosis.Conclusion: Imprint smear technique is a simple, rapid, inexpensive and reliable procedure. The routine use of imprint smear in the bone marrow examination will serve as an invaluable adjunct to bone marrow aspiration and biopsy.Journal of Pathology of Nepal (2015) Vol. 5, 739-746

Mature B Cell Acute Lymphoblastic Leukemia Presenting with Hypercalcemia

Hypercalcemia is rarely observed in acute lymphoblastic leukemia. So far, all cases presenting with hypercalcemia are pre-B cell ALL. In this case, a mature B-cell ALL patient presenting with hypercalcemia is discussed. A threeyear-old boy had a history of fever, weakness, swelling and pain in both kness. In the patient’s blood smear, 93% L3 type blasts were seen and 90% L3 type large blasts with vacuoles were seen in the bone marrow aspiration smear. The flow cytometry results were as follows: CD10: 87%; CD19: 85%; KAPPA: 66%; and Lambda: 35% was compliant with mature B cell ALL. His calcium level: 15 mg/dl. Although previous cases imply that hypercalcemia is usually treated with pamidronate, calcium levels gradually decreased to normal levels within five days with intravenous fluid therapy, furosemide, and steroids in our case. Conclusion: Hypercalcemia is rare in leukemia. So far, all cases presenting with hypercalcemia are pre-B cell ALL. It is important to know that hypercalcemia could be seen in the mature B-cell ALL.

Bone-marrow spectrum in a tertiary care hospital: Clinical indications, peripheral smear correlation and diagnostic value

Aim: The aim was to identify the indications for a bone-marrow aspiration study, to correlate between peripheral blood smear findings and those of bone-marrow aspiration; and to assess the diagnostic value of bone-marrow aspiration examination. Settings and Design: A retrospective study was done on patients referred for bone-marrow aspiration in a tertiary care hospital for a period of about 3 years. Materials and Methods: Stained bone-marrow aspirate smears were examined. Records regarding the clinical indication for the procedure, peripheral blood smear reports, blood counts and significant findings on bone-marrow aspiration examination were retrieved. The role of bone-marrow aspiration in the diagnosis of hematological and nonhematological disorders was reviewed in the study. Results: Of a total of 153 cases, 24 (15.68%) were from the pediatric age group and 129 (84.32%) were adults. Male:female ratio was 1.3:1. The indications were anemia for evaluation (33%), evaluation of pancytopenia (26%), suspicion of malignancy (17%), fever (10%) and thrombocytopenia (3%). Bone-marrow aspiration yielded a diagnosis of hematological malignancy in a total of 11 cases. However, peripheral blood smear examination was able to pick up only seven of these cases. Furthermore, one case of metastatic deposits was seen in a case of carcinoma of unknown primary. The bone-marrow findings correlated with the peripheral blood smear findings in 54 (43.2%) out of 125 of the total cases. Conclusion: This study shows that bone-marrow aspiration is a valuable diagnostic tool and aids in diagnoses and etiology of various hematological findings such as anemia and pancytopenia.

Central Morphology Review of Childhood Bone Marrow Failure in Japan

In the diagnosis of childhood bone marrow failure (BMF), differentiating aplastic anemia (AA) from hypoplastic myelodysplastic syndrome (MDS) is challenging. In addition, inherited BMF (IBMF) should be excluded from acquired BMF. The 2008 WHO classification has proposed a provisional entitiy, “refractory cytopenia of childhood (RCC)”. The spectrum of patients with RCC is wide, ranging from patients with severe hypocellular bone marrow (BM) and mild dysplasia to those with normocellular BM and distinct dysplasia meeting the criteria for refractory cytopenia with multilineage dysplasia (RCMD) in adults. Currently, it is recommended that children who meet the criteria for RCMD should be considered as RCC in the WHO classification until the number of lineages involved have been fully evaluated with regard to their relative importance as prognostic factors in childhood MDS.To enable diagnosis based on the WHO classification, the Japanese Society of Pediatric Hematology and Oncology in February 2009 established a central review system of BM morphology, including peripheral blood (PB) and BM smears and specimens from trephine biopsies in childhood BMF. PB and BM smears were reviewed by two pediatric hematologists, and the specimens from BM trephine biopsies were reviewed by a pathologist. In addition, the telomere length of lymphocytes and paroxysmal nocturnal hemoglobinuria (PNH) clones in PB were measured by flowcytometry for patients with BMF. RCC is defined as persistent cytopenia with <2% and <5% blasts in PB and BM, respectively. BM aspirate smears show dysplastic changes in >2 cell lineages or >10% within one cell lineage. On the other hand, the criteria of RCMD in adult MDS is defined as persistent cytopenia with <1% and <5% blasts in PB and BM, respectively. BM smears show >10% dysplastic changes in >2 cell lineages. We introduced the RCMD criteria in this central review.From February 2009 to October 2013, 1,000 cases including 536 males and 464 females were prospectively reviewed. The median age was six years (range, 0–39 years). Of the 1,000 cases, 575 were classified as BMF, and of them, 137 were classified as AA, 236 as RCC, 103 as RCMD, 38 as hepatitis-related BMF, 3 as PNH and 58 as IBMF. Of the 58 cases with IBMF, 21 were diagnosed as Fanconi anemia, 12 as Shwachman–Diamond syndrome, and 8 as dyskeratosis congenita. Seventeen patients suspected of IBMF were undiagnosed. In 97 advanced cases of MDS, 24 were classified as refractory anemia with excess blasts (RAEB), 6 as secondary MDS, and 21 as therapy-related MDS. To determine the clinical differences among AA, RCC, and RCMD, we compared laboratory and clinical data for 476 patients classified as AA, RCC, and RCMD. Median ages in the AA, RCC, and RCMD groups were 9, 8, and 7 years, respectively (p = 0.007). The male/female ratio in AA, RCC, and RCMD groups was 1.1, 1.2, and 3.6, respectively (p = 0.034). When patients were classified according to the disease severity criteria for AA, 78% of the patients with AA had very severe or severe disease, whereas only 38% of the patients with RCC and 28% of the patients with RCMD had very severe or severe disease (p < 0.001). Chromosomal abnormalities were detected in two patients (1%) with AA (trisomy 8), 10 patients (4%) with RCC (monosomy 7, n = 2; trisomy 8, n = 6; other, n = 2), and 12 patients (12%) with RCMD (monosomy 7, n = 5; trisomy 8, n = 2; other, n = 5) (p = 0.001). Out of the 476 patients, 67 (AA, n = 32; RCC, n = 32; RCMD, n = 3) were administered IST with rabbit antithymocyte globulin (ATG) and cyclosporine. After 6 months, the response rate to IST was not significantly different among the three groups; AA, 41%; RCC, 47%; RCMD, 100% (p = 0.142).In conclusion, the entity of RCMD should be applied to childhood MDS because patients with RCMD exhibited a significantly high frequency of chromosomal abnormalities at the time of diagnosis. To definitively determine whether these three diseases are different entities, it would be necessary to prospectively compare the clinical outcomes and biological findings in a larger number of patients with AA, RCC, and RCMD. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Involvement in Neuroblastoma: A Study of Hemato-morphological Features.

Bone marrow involvement in neuroblastoma indicates advanced stage of disease. The recent use of autologous bone marrow "rescue", has provided an additional important reason for accurate assessment of bone marrow status in newly diagnosed patients. In this study, we analyzed 44 cases of neuroblastoma for bone marrow infiltration status and their hematological parameters. Eighty-eight bone marrow aspirate and trephine touch imprint smears and 44 trephine biopsy sections were examined in these 44 patients. Of these, 24 cases (54.5%) showed marrow infiltration. Leucopenia and bicytopenia were significantly (p<0.05) associated with marrow infiltration. Both bone marrow aspirate and biopsy were positive for infiltration in 16 out of 24 positive cases. Only aspirate smears were positive in 4 and only trephine biopsy in another 4 cases. The pattern of infiltration consisted of rosette formation in 40.7% cases on aspirate smears and 22.2% cases in trephine biopsies. Remaining cases showed diffuse and interstitial presence of tumor cells and cases positive only on trephine biopsy, showed marked stromal reaction. Bilateral trephine biopsies combined with aspirate smears picked up all positive cases compared to when they were assessed alone.

Co-occurence of Visceral Leishmaniasis and Lymphoma

Clinicians have usually considered malignancies during follow up of patients who have infectious diseases as a pre-diagnosis. However, malignancy and an infectious disease are seen together more rarely, with the exception of immunosuppressed patients. This presentation is a case report followed up for fever of unknown origin. The patient was admitted to the hospital with the symptoms of fever, weight loss, abdominal pain and weakness. Anemia and hypergamaglobulinemia by biochemical analyses and splenomegaly by total body computed tomography were detected. Amastigotes were seen in bone marrow aspiration smears and promastigotes were isolated in NNN medium. At the end of the Liposomal Amphotericin B treatment, control bone marrow aspiration was applied. Leishmania amastigotes were not seen, while patient was diagnosed as diffuse B cell lymphoma pathologically.

Floatation of decalcified bone marrow core biopsy - a clue to marrow hypocellularity.

Background : Examination of bone marrow plays a pivotal role in the practice of haematology. It can be evaluated by three ways - bone marrow aspiration smears (BMA), bone marrow touch imprints (BMI) and bone marrow biopsy (BMB). BMB sections are considered to be the gold standard for assessing overall marrow cellularity. To evaluate the correlation, if any, between bone marrow cellularity and floatation pattern of the core biopsy specimen, after proper decalcification. This study was carried out in the Department of Pathology, Institute of Medical Sciences, Varanasi over a period of 26 months. Specimens of BMA, BMI and BMB were collected from 182 cases. The core biopsy specimens were fixed in 10% buffered formalin for 24 hours, and were decalcified in 5% formic acid for 12 hours. The properly decalcified core biopsy samples were then put into adequate-sized container filled with 10% buffered formalin, and floatation pattern was documented. All the observations were evaluated using simple and basic statistical tools, i.e. sensitivity, specificity, positive predictive value. Chi square test was applied for obtaining statistical correlation i.e. p-value. Out of 182 core biopsy specimens, 32.4% (n=59) floated, while rests sank. Out of the 59 floating core biopsies, 57 were hypocellular. Seven core biopsies, among 123 specimens that sank, were hypocellular. The sensitivity and specificity of floatation pattern for hypocellular marrow were 89.2% and 99.1%, respectively. A strong correlation (p-value <0.001) between the floatation pattern and bone marrow cellularity was obtained. Assessment of floatation pattern of properly decalcified marrow core specimen is reliable for assessing marrow hypocellularity.

Human leucocytic antigen-DR negative acute myeloid leukemia: A diagnostic dilemma for hematopathologist

Background: Acute myeloid leukemia (AML) blast variably express Human leucocytic antigen (HLA).We retrospectively analyzed immunophenotypic and clinical profile of 12 cases of HLA -DR negative AML and correlated with their morphological, cytogenetics and Molecular findings.There is a paucity of literature mentioning morphological, immunophenotypic and cytogenetics characteristics of HLA DR negative AML. Aim: This study was designed to study the morphological, flow cytometric, and cytogenetics characteristics of HLA DR negative AML/non acute Promyelocytic Leukaemia (APML) cases. Materials and Methods: Seventeen such cases were diagnosed over a period of 1 year and 8 months. Peripheral blood and bone marrow aspiration smears were stained by Wright giemsa and examined by three hematopathologist independently. Immunophenotyping was done using multicolour flow cytometry on BD FACS CANTO II using FACS DIVA software.Conventional Karyotyping was done using Wright giemsa staining (using IKAROS software) and florescent in situ hybridization (FISH) was done using dual color dual fusion probe from Vysis promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) fusion gene probe. Molecular analysis using reverse transcriptase-polymerase chain reaction (RT-PCR) was done using Thermal Cycler of Applied Biosystem and Gel-Doc by Biorad. Results : Of the 12 cases studied ten were classified as French-American-British (FAB) AML-M1. Two case as FAB AML-M2. Morphologically the cells resemble abnormal promyelocytes with bilobation, convoluted and folded nucleus, inconspicuous nucleoli and open chromatin (n = 11) and with blastic morphology, open chromatin, and inconspicuous nucleoli (n = 1).Karyotyping analysis shows normal karyotype (n = 10), del 9q-(n = 1), and t (5:9) (n = 1) respectively.FISH done using dual color dual fusion probe (n = 12) do not show PML-RARA fusion signal.RT-PCR (n = 12) revealed a negative result for PML - RARA fusion transcripts. Conclusion: HLA-DR negativity does not always imply a diagnosis of APML. A Cytogenetic (FISH/conventional karyotype) or molecular (RT-PCR) evidence of t (15:17) or PML-RARA fusion gene transcript is a must to stamp a case as APML. Morphology and Flow cytometric findings are only complementary to Cytogenetic/Molecular findings.

Peripheral blood smears, bone marrow aspiration, trephine and clot biopsies: methods and protocols.

Maximum diagnostic information is obtained when peripheral blood smears, bone marrow aspiration smears, trephine biopsy imprints, trephine and clot biopsy sections are simultaneously examined. Peripheral blood smears reflect end organ function and provide clues to underlying hematolymphoid pathology that may prompt additional studies including bone marrow examination. Bone marrow aspiration alone has diagnostic utility in the evaluation of a limited number of primary hematological conditions including: megaloblastic anemias, hyporegenerative anemias, certain hemolytic anemias, normochromic normocytic anemias, neutropenias, thrombocytopenias, immunoglobulin disorders, storage diseases, and leukemias (Bain, J Clin Pathol 54:657-663, 2001). Bone marrow trephine biopsy is indicated in those situations where marrow aspiration is unsuccessful; in evaluation of cytopenias, myelofibrosis, suspicion of lymphoma, metastatic tumor, granulomatous disease, evaluation of myeloproliferative neoplasms, and for the examination of trabecular bone in metabolic diseases (Bain, J Clin Pathol 54:737-742, 2001). Many of the indications for marrow aspiration overlap with those for trephine biopsy. Because it is not possible to predict which patients will have diagnostic aspiration biopsies and which will have diagnostic trephine biopsies, both procedures are routinely performed together (Brynes et al., Am J Clin Pathol 70:753-759, 1978; Cotelingam, Adv Anat Pathol 10:8-26, 2003; Lee et al., Int J Lab Hematol 30:349-364, 2008; Peterson et al., Arch Pathol Lab Med 126:1050-1056, 2002).