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Bone marrow biopsy depends on tissue morphology, immunohistochemical staining, and moleculardetection. Tissue pretreatment is required for bone marrow samples, from clinical specimen acquisition to pathological reporting, but during the process, proteins and nucleic acids are often altered because of the acid in fixation and decalcification solutions. In our study, we present an easy and effective pretreatment protocol and compared this novel pretreatment protocol (Set 2) with an existing traditional pretreatment process (Set 1) using tissue morphology, IHC staining, and molecular pathological analyses. Granulocytic IHC markers showed more intensive staining in samples of Set 2 than in those of Set 1. The Set 2 protocol provided a higher DNA yield and less fragmentation; moreover, samples processed with the Set 2 protocol could be subsequently used in FISH and DNA sequencing assays. Our optimized novel pretreatment protocol could better protect proteins and DNA molecules while maintaining good cell morphology compared to traditional pretreatment The novel pretreatment reagents could role as a reference by more laboratories for pretreating bone marrow biopsy samples and scientific research.

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Bone marrow aspiration and trephine biopsies are commonly used procedures in clinical practice. The practice of making a clot section by using the leftover blood from the bone marrow aspirate material is not a commonly followed practice across centers. A clot section has the advantage of studying the added material with an increased possibility of detecting focal lesions such as myeloma, lymphoma, granuloma, and metastasis in the bone marrow. Bone marrow aspirate, trephine biopsy, and clot section were compared for the detection of focal lesions in a series of 5 patients, 3 of who presented with a history of fever and 2 were already diagnosed cases of Hodgkin lymphoma. Focal lesions were detected in the 5 cases in the clot section alone, whereas bone marrow aspirate and trephine biopsy did not show any focal lesion. Granulomatous infiltration was detected in 3 patients, and lymphomatous infiltration was detected in 2 patients in the clot section, whereas bone marrow aspirate and trephine biopsy were negative for any focal lesion in all 5 cases. A clot section is particularly useful in the detection of bone marrow lesions with a focal distribution. Hence, it must be studied alongside bone marrow aspirate smears, touch smears, and trephine biopsy to increase the diagnostic yield.

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Nucleophosmin (NPM1) mutation is one of the most common recurring genetic abnormalities seen in acute myeloid leukemia (AML). Immunohistochemistry serves as a cost effective and simple surrogate testing method for detection of NPM1 mutation. This study was conducted to evaluate the frequency of aberrant cytoplasmic nucleophosmin 1 expression in leukemic blast cells on formalin fixed bone marrow trephine biopsy (BMB) sections and also to correlate this data with the reference molecular method (reverse transcriptase-polymerase chain reaction; RT-PCR and gene sequencing), where available. Immunostains were performed using mouse anti-NPM1 monoclonal antibody on 71 paraffin embedded bone marrow biopsies (BMB) of patients with AML of any French-American-British (FAB) subtype. Results of immunohistochemistry (IHC) were then compared with the reference molecular method. The proportion of NPM1 expression by immunostaining in AML cases was found to be 17%. Twelve of the total 71 cases demonstrated cytoplasmic nucleophosmin (NPMc+) on immunostaining. Eleven of the positive cases that were correlated with the molecular standard demonstrated mutation in exon 12 of NPM1 gene. Cytoplasmic nucleophosmin expression by immunostaining was found to be in complete agreement with the standard molecular method. In a resource restricted setup, the information from this study might help in providing an inexpensive and accurate detection method to facilitate introduction of this marker in diagnostic and prognostic workup of AML especially in patients showing normal karyotype and no common recurrent translocations.

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OBJECTIVE: To determine the aetiologies of pancytopenia based on bone trephine biopsy among paediatric and adult patients. Method: The retrospective cross-sectional study was conducted at the Haematology Department of Aga Khan University Hospital, Karachi, and comprised data from June 1, 2016, to October 31, 2019 related to pancytopenia patients who underwent bone marrow biopsy. Data included age, gender, presenting symptoms, physical examination, complete blood count, peripheral smear, bone marrow aspirate and trephine biopsy findings and final diagnosis. Data was analysed using SPSS 19. RESULTS: Of the 2852bone marrow biopsies done, 255(9%) related to evaluation of pancytopenia. Of them, 208(82%) were adult and 47(18%) were paediatric patients. The median age for adults was 38.8 years (range: 16-92years) and that in paediatric patients was 10.9 years (range: 2-15 years). Presenting symptoms were available for 182(71.4%) patients, and the commonest symptom was generalised weakness 128(70.3%). Overall, pallor was the most frequent sign 233(93.2%). Anisocytosis was predominant blood smear finding 156(61.1%), while the commonest aetiology was aplastic anaemia in both paediatric 23(49%) and adult 57(27.4%) groups. Bone marrow biopsy established the diagnosis in 253(99.2%) cases, while 2(0.95%) adult cases were not diagnosed. Of the diagnosed cases, 103(40.4%) were malignant; 15(32%) paediatric patients and 88(42.3%) adults. The rest were benign; 31(67.4%) paediatric patients and 119(3%) adults. CONCLUSIONS: Bone marrow biopsy helped in diagnosing all but 2 pancytopenic patients. Aplastic anaemia was the commonest cause in both paediatric and adult patients.

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We evaluated the impact of bone marrow sample characteristics on the detection of persistent cytogenetic abnormalities (PCA) following induction chemotherapy for acute myeloid leukemia (AML). PCA's were identified in 20.4% of patients and were more common with complete remission without count recovery (CRi) vs. those with count recovery (CR, 45.8 vs. 13.5%, p = .001), with >2% blasts vs. ≤2% blasts (42 vs. 12%, p = .001) and with hypocellular trephine biopsies relative to those with normo/hypercellular biopsies (42.1 vs. 17.3%, p = .03), although in a multivariate analysis only CRi and blast count >2% were independently associated with a PCA. PCA's were not observed in patients with favorable risk karyotype. Amongst patients with intermediate and unfavorable risk karyotypes PCA were not associated with differences in overall or, amongst non-transplanted patients, relapse free survival. Thus, although PCAs are common post-induction it is unclear whether they provide any independent prognostic information beyond the diagnostic karyotype.

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BACKGROUND AND OBJECTIVES: To compare bone marrow biopsy (BMB) and PET/CT in detecting bone marrow involvement in Hodgkin's lymphoma MATERIAL AND METHODS: Retrospective analysis of 65 patients with both tests in the initial staging or in relapse with special attention to the PET/CT uptake pattern. RESULTS: In 3 patients (4.6%), the BMB showed bone marrow involvement with the PET/CT being positive in them all: 2 with diffuse+multifocal pattern and one diffuse only. In 11 additional patients (total 14/65, 21%), bone marrow involvement was diagnosed by PET/CT because bone marrow uptake was above hepatic one. The pattern was focal only in 2 cases, multifocal in 5, diffuse in 3 and diffuse+multifocal in one. In these last 4 cases the BMB showed an unspecific myelopathy. CONCLUSIONS: PET/CT detects all cases with BMB affected and many that escape to biopsy, however when the uptake pattern is diffuse it could be by involvement or reactive hyperplasia and in those cases the BMB should be done.

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Most guidelines suggest that only the bone marrow aspirate (BMA) is necessary to assess residual disease following intensive chemotherapy for Acute Myeloid Leukemia (AML) with the bone marrow trephine biopsy (BMTB) recommended in cases of a poor quality BMA. We performed a retrospective study evaluating this in a cohort of patients receiving intensive chemotherapy for AML. Residual disease was assessed by morphological examination of the BMA and BMTB±immunohistochemistry. Of the 647 marrows 32.6% were interim marrows performed prior to peripheral count recovery, 41.7% were end of induction (EOI) marrows and the remaining were 'other marrows'. The BMA and BMTB findings were concordant in 92.8% of cases. The BMTB led to a change in diagnosis from 'no leukemia' to 'residual leukemia' in 5.2% of interim, 3.7% of EOI and 2.4% of 'other' marrows. The BMA alone had a sensitivity of 86.8% in detecting residual leukemia and of 82.3%, 82.5% and 94.2% for interim, EOI and 'other marrows', respectively. Despite the high concordance between the BMA and the BMTB the poor sensitivity of the BMA in detecting residual leukemia, particularly at EOI, may lead to an overestimation of the complete remission rates which may have therapeutic and clinical trial implications.

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This study aimed to document the morphological and immunophenotypic features, and describe the diagnostic features of bone marrow (BM) involvement in human herpes virus 8 Multicentric Castleman disease (HHV8-MCD). BM trephine biopsy (BMTB) specimens from 28 patients were revisited. Samples were evaluated for expression of CD3, CD20, CD138, CD68R, glycophorin C, CD42b, HHV8-latency-associated nuclear antigen (LANA1), Epstein-Barr virus-encoded small RNA and light chains. Presence of significant numbers of HHV8-LANA1(+) lymphoid/plasmacytic cells, noted in 10/28 cases, was indicative of BM involvement and was associated with low CD4 and CD8 counts in peripheral blood. The characteristic morphological appearance of MCD seen in lymph nodes is a rare finding in BMTB. 4/5 cases with lymphoid aggregates were involved by MCD, whereas 6/23 cases without lymphoid aggregates were involved by MCD (P = 0·023). 9/18 cases with hypercellular marrow were involved by MCD, whilst only 1/8 cases with normo/hypocellular marrow showed involvement by MCD (P = 0·070). While 9/21 cases with increased marrow reticulin were involved by MCD, none of the cases with no increase in reticulin were involved by MCD (P = 0·080). Reactive plasmacytosis is a frequent finding. We conclude that bone marrow is involved in a significant proportion of patients with MCD (36%), and involvement can be identified by HHV8-LANA1 immunohistochemistry.

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OBJECTIVES: Bone marrow (BM) aspiration and trephine biopsy is one of the most valuable procedures in the evaluation of hematological disorders. There is a shortage of published literature regarding the indications, procedure, and outcome of bone marrow examination (BME) in neonates and infants. The aim of the present study is to analyze the common indications of performing BME and to assess the spectrum of disorders diagnosed from BM of neonates and infants. METHODS: A retrospective analysis of BMEs performed in infants over a period of 5 years, between 2009 and 2013 was done. RESULTS AND DISCUSSION: A total of 297 BME were performed on 285 infants, which constitutes 10.3% of pediatric BME procedures during the same period. In our institute, BME is routinely performed by trained pathologists from posterior superior iliac spine in children including infants and neonates with an overall sample adequacy of 97%. Evaluation of cytopenias and suspicion of storage disorder were the most common indications for BME procedure, while acute leukemias and storage disorders were the most common diagnoses offered in infant BM. CONCLUSIONS: Posterior superior iliac spine is a good site of BME in neonates and infants. BM trephine biopsy is a difficult procedure in this age group, however remains indispensable in situations where an infiltrative pathology is suspected. BME not only helps to make specific diagnoses but should also be used as an extremely valuable, quick, and economically viable procedure to exclude major hematological disorders including certain forms of storage disorder and hematological malignancy in this age group.

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BACKGROUND: The bone marrow is frequently involved in variety of cases presenting with hematological and non-hematological disorders, which are diagnosed by two separate but interrelated techniques such as bone marrow aspiration (BMA) and bone marrow biopsy (BMB). AIM: This study was aimed to assess the diagnostic value of the BMA and BMB and role of both the procedures to reach final diagnosis when done simultaneously. SETTINGS AND DESIGN: It was a prospective study. The findings of BMA smears were correlated with BMB sections and data obtained was analysed. MATERIALS AND METHODS: BMA and BMB were performed on 50 patients. Criteria of inclusion included the main indications for performing this procedure, the availability of full medical records and patient consent. The patients had a male to female sex ratio of 1.6:1 and a wide age range from 4 years to 74 years. RESULTS: In the present study, the main indications for bone marrow examination were categorized. Out of 50 cases studied, in 23 cases, a strong positive correlation between BMA and BMB was noted. However, it was found that in the cases of aplastic anaemia, different phases of myeloproliferative neoplasm (MPN), multiple myeloma, tubercular granulomas and hemato-lymphoid neoplasm, involvement of the marrow was detected better in bone marrow biopsies. CONCLUSION: The study concludes that preparations of aspirate and trephine biopsy are easy, rapid and complementary to each other in majority of the lesions. The advantage of both the procedures done together enabled us to study the cytomorphology of the cells along with the pattern of distribution of the cells depending on the cases, hence help in making the diagnosis accurately.

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AIMS: The diagnosis of primary myelofibrosis (PMF) strongly relies on the bone marrow biopsy findings, but a report model has not been standardised. Our aim was to establish general recommendations for bone marrow evaluation and standardised reporting in a case suspicious of PMF. METHODS: The Delphi method was employed to obtain expert consensus. An advisory panel of 10 leading members identifies a total of 37 haematopathology experts to participate. The first Delphi round included a questionnaire with three main groups of items: minimal clinical and laboratory data considered necessary before reporting, minimal descriptive aspects to record and main histological differential diagnosis. The final report content was based on consensus obtained after the second Delphi round. RESULTS: The minimal data considered necessary were age, splenomegaly, haemoglobin, leucocyte and platelet counts, differential blood cell count, leucoerythroblastic blood picture, lactate dehydrogenase (LDH) level, BCR-ABL and JAK2 mutational status, reticulin stain and the internal control for the reticulin staining. The minimal descriptive aspects to report were cellularity, osteosclerosis, megakaryocytic morphology and localisation, dense megakaryocytic clusters, quantity of granulocytic precursors, grade of myelofibrosis in a scale of 4, and a proposed final diagnostic approach. The entities to be considered for differential diagnosis were mainly the other classical chronic myeloproliferative neoplasms. CONCLUSIONS: The Delphi method is a robust tool to determine essential information to be included in a pathology report. A standardised good-quality histopathological report form may help to homogenise PMF diagnosis. A close collaboration between the pathologist and the haematologist is desirable according to our survey.

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PATIENT: Male, 19 FINAL DIAGNOSIS: Hyperleukocytosis • thrombocytosis SYMPTOMS: Hyperleukocytosis • retroperitoneal hemorrhage • thrombocytosis MEDICATION: - Clinical Procedure: Bone marrow trephine biopsy Specialty: Hematology • Radiology. OBJECTIVE: Diagnostic/therapeutic accidents. BACKGROUND: Bone marrow (BM) trephine biopsy is generally a safe procedure, but adverse events such as retroperitoneal hemorrhage (RPH) may occur. We report 3 cases of this complication. CASE REPORT: A 19-year-old male with thrombocytopenia and coagulopathy underwent BM trephine biopsy to confirm relapse of acute lymphoblastic leukemia. Two hours later, he developed severe hypotension and a CT scan revealed a massive RPH, and was treated conservatively. The RPH recurred 2 weeks after chemotherapy and was successfully treated with gel foam embolization. A 55-year-old male with coagulopathy underwent BM trephine biopsy for hyperleukocytosis and thrombocytosis. He developed a large RPH preceded by left lumbar dermatome sensory neuropathy. He was treated conservatively. A 56-year-old overweight woman on aspirin underwent BM trephine biopsy for polycythemia. Twelve hours later she developed severe abdominal pain with hypotension. A CT scan showed a massive RPH and secondary hemothorax. She was treated conservatively and the RPH resolved after several months. CONCLUSIONS: We and others showed that myeloproliferative neoplasm, quantitative or qualitative platelet abnormalities, aspirin, coagulopathy, and obesity are associated with development of RPH following BM trephine biopsy. Early diagnosis and intervention are crucial. Correction of coagulopathy and cessation of anti-platelet treatment prior to biopsy can prevent this serious complication.