Digital assessment of peripheral blood and bone marrow aspirate smears.

The diagnosis of benign and neoplastic hematologic disorders relies on analysis of peripheral blood and bone marrow aspirate smears. As demonstrated by the widespread laboratory adoption of hematology analyzers for automated assessment of peripheral blood, digital analysis of these samples provides many significant benefits compared to relying solely on manual review. Nonetheless, analogous instruments for digital bone marrow aspirate smear assessment have yet to be clinically implemented. In this review, we first provide a historical overview detailing the implementation of hematology analyzers for digital peripheral blood assessment in the clinical laboratory, including the improvements in accuracy, scope, and throughput of current instruments over prior generations. We also describe recent research in digital peripheral blood assessment, particularly in the development of advanced machine learning models that may soon be incorporated into commercial instruments. Next, we provide an overview of recent research in digital assessment of bone marrow aspirate smears and how these approaches could soon lead to development and clinical adoption of instrumentation for automated bone marrow aspirate smear analysis. Finally, we describe the relative advantages and provide our vision for the future of digital assessment of peripheral blood and bone marrow aspirate smears, including what improvements we can soon expect in the hematology laboratory.

Childhood-onset systemic lupus erythematosus with trisomy X and the increased risk for bone complications: a case report.

BACKGROUND: Systemic lupus erythematosus is a multi-organ inflammatory autoimmune disease; immune complexes are part of the pathogenesis, but not entirely responsible. Trisomy X is the most common female chromosomal abnormality and the role of an additional X chromosome in the development of systemic lupus erythematosus is well recognized. However, the potential complications and optimal management of childhood lupus with trisomy X remain unclear. Herein, we describe a case of childhood-onset systemic lupus erythematosus associated with severe bone complications presumably secondary to trisomy X. CASE PRESENTATION: A 16-year-old Japanese girl was diagnosed with childhood-onset systemic lupus erythematosus and trisomy X. A chromosomal abnormality (47, XXX) was incidentally identified on bone marrow examination initially done to determine the cause of pancytopenia. She had a persistent headache, fever　for six days, diffuse hair loss, mucosal ulcers, butterfly eruptions, and palmar erythema. Furthermore, thrombocytopenia, anemia, and erythrocyte fragmentation were detected, suggesting secondary thrombotic microangiopathy. She was initially treated with intravenous methylprednisolone pulse therapy and prescribed monthly cyclophosphamide for severe disease activity, prednisolone, mycophenolate mofetil, and hydroxychloroquine as remission maintenance drugs. She developed generalized extremity pain that had been worsening throughout the disease. Extremity magnetic resonance imaging performed 12 months after the treatment onset revealed multifocal avascular necrosis, and dual-energy X-ray absorptiometry revealed further decreased bone mineral density. High plasma levels of factor VIII were detected by additional tests for coagulation functions, and we suspected the possibility that factor VIII might cause avascular necrosis due to thrombosis. Currently, she is being treated with prednisolone and MMF for SLE. However, her extremity pain has not been managed effectively even under the administration of non-steroidal anti-inflammatory drugs and pregabalin. CONCLUSIONS: An additional X chromosome has been reported to be associated with factor VIII and osteoporosis. Additionally, elevated plasma levels of FVIII is the risk factors for thrombosis, which leads to the risk of avascular necrosis. Patients with systemic lupus erythematosus complicated by trisomy X might be at a higher risk of avascular necrosis and osteoporosis that can also manifest in childhood systemic lupus erythematosus.

Whole body FDG-PET/CT for the assessment of bone marrow infiltration in patients with newly diagnosed lymphoma.

BACKGROUND: Positron emission tomography-computed tomography (PET-CT) and bone marrow biopsy are currently the common clinical examination of lymphoma infiltration. The aim of this research is to evaluate the value of PET-CT in diagnosis of bone marrow infiltration, clinical staging and pathological typing of lymphoma. METHODS: 153 cases were analyzed retrospectively to compare the consistency of PET-CT and bone marrow biopsy. We analyzed the sensitivity, accuracy and specificity of PET-CT in different clinical pathology of lymphoma. RESULTS: The PET-CT sensitivity in detecting bone marrow infiltration is 54.3% with a specificity of 80.5% and accuracy of 74.5%. In aggressive B-cell lymphoma (DLBCL, HG-BL) and MZL, PET-CT results of bone marrow infiltration showed high accuracy of 88.1% and 83.3% respectively. The median value of SUVmax in the patients detected to have bone marrow infiltration by BMB was significantly higher than patients with BMB negative results among subgroups of aggressive B-cell lymphoma, MZL and T-NHL (p<.05). CONCLUSION: PET-CT is significant in detecting bone marrow infiltration in certain pathological types of lymphoma. However pathological inconsistencies still exist between bone marrow biopsy and PET-CT, thus PET-CT cannot completely replace biopsy.

Comparison of methodologies for the detection of BRAF mutations in bone marrow trephine specimens.

AIMS: BRAF V600E detection assists in the diagnosis of hairy cell leukaemia (HCL); however, testing practices vary. We evaluated the clinical utility of 5 BRAF mutation testing strategies for use on bone marrow trephines (BMT). METHODS: 11 HCL, 5 HCL 'mimic', 2 treated HCL and 10 normal BMT specimens were tested for mutant BRAF, comparing Sanger sequencing, pyrosequencing, amplicon-based next generation sequencing (NGS), automated (Idylla) PCR and immunohistochemistry (IHC). RESULTS: PCR and IHC were cheaper and identified V600E in 100 % of HCL cases. Pyrosequencing detected the mutation in 91%, NGS in 55% of cases and Sanger sequencing in 27%. All assays gave wild-type BRAF results in HCL mimics and normal BMT samples. CONCLUSIONS: PCR and IHC were most sensitive and cost-effective, but these have limited scope for multiplexing and are likely to be replaced by NGS gene panels or whole genome sequencing in the medium to long term.

Bone marrow core biopsy in 508 consecutive patients with chronic myeloid leukemia: Assessment of potential value.

BACKGROUND: The diagnosis of chronic myeloid leukemia (CML) is based on characteristic clinical and laboratory findings and the presence of BCR/ABL1 in the blood and/or bone marrow (BM). The utility of BM core biopsy in the workup of patients with CML has been questioned. METHODS: The potential added value of BM biopsy versus aspiration in the workup of a single-institution series of 508 patients with CML at their initial presentation was systematically assessed. BM biopsy was considered essential when it was needed to establish the disease phase, often because blast counts derived from aspirate smears were misleading because the biopsy specimen was more representative of the disease. BM biopsy was considered helpful if it was needed for other nonessential reasons. RESULTS: In 127 patients (25%), BM biopsy was either essential (109 patients) or helpful (18 patients). Patients with accelerated-phase (AP) or blast-phase (BP) disease often required a biopsy related to essential reasons. High-grade myelofibrosis (MF) was more frequent in patients with AP/BP disease than patients with chronic-phase disease (P = .0005), and the identification of BP disease required a BM biopsy assessment in 75% of the patients (P = .001). A follow-up BM evaluation more often yielded inadequate aspirates in patients with inadequate BM aspirates at the time of their initial diagnosis. CONCLUSIONS: BM core biopsy remains valuable in the workup of 25% of patients with CML because it facilitates identification of the disease phase or MF. The initial grade of MF is associated with the disease stage and outcome after therapy. BM biopsy is, therefore, indicated for patients with CML who have AP/BP disease or other findings suggestive of progressive disease.

Flow cytometric minimal residual disease assessment of peripheral blood in acute lymphoblastic leukaemia patients has potential for early detection of relapsed extramedullary disease.

OBJECTIVES: To evaluate peripheral blood (PB) for minimal residual disease (MRD) assessment in adults with acute lymphoblastic leukaemia (ALL). METHODS: We analysed 76 matched bone marrow (BM) aspirate and PB specimens independently for the presence of ALL MRD by six-colour flow cytometry (FC). RESULTS: The overall rate of BM MRD-positivity was 24% (18/76) and PB was also MRD-positive in 22% (4/18) of BM-positive cases. We identified two cases with evidence of leukaemic cells in PB at the time of the extramedullary relapse that were interpreted as MRD-negative in BM. CONCLUSIONS: The use of PB MRD as a non-invasive method for monitoring of systemic relapse may have added clinical and diagnostic value in patients with high risk of extramedullary disease.

Bone marrow solid core biopsy needle: a critical assessment of the utility, benefits and limitations of the instruments employed in current day haematology and oncology.

The optimal clinical evaluation of the bone marrow requires an examination of air-dried and well-stained films of the aspirated tissue along with a histopathological evaluation of adequately processed and properly stained core biopsy specimens. A bone marrow evaluation can be essential in establishing a diagnosis, determining the efficacy of treatment in haematological disorders and to monitor haematological status of patients following bone marrow/stem cell transplantation. It is also an essential component of the staging process for newly diagnosed malignancies. Currently available bone marrow aspiration needles are quite satisfactory and if properly used provide good-quality specimens for morphological evaluation. However, if a bone marrow core biopsy is concerned, several needles are currently in use but not all of them provide good-quality biopsy specimens for histological evaluation or are user friendly. We have compared the recently introduced Moeller Medical single use bone marrow core biopsy needle with the Jamshidi needle with marrow acquisition cradle (CareFusion), J-needle (Cardinal Health) and OnControl device (Vidacare). It is concluded that the Moeller Medical needle system has definite advantages over others and is recommended for routine use.

Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group.

BACKGROUND: The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. METHODS: A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. RESULTS: To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. CONCLUSIONS: The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate collaborative trial design. Cancer 2017;123:1095-1105. © 2016 American Cancer Society.

Assessment of bone marrow involvement in patients with lymphoma: report on a consensus meeting of the Korean Society of Hematology Lymphoma Working Party.

In September 2011, the Korean Society of Hematology Lymphoma Working Party held a nationwide conference to establish a consensus for assessing bone marrow (BM) involvement in patients with lymphoma. At this conference, many clinicians, hematopathologists, and diagnostic hematologists discussed various topics for a uniform consensus in the evaluation process to determine whether the BM is involved. Now that the discussion has matured sufficiently to be published, we herein describe the consensus reached and limitations in current methods for assessing BM involvement in patients with lymphoma.

Comparative Assessment of Vitamin-B12, Folic Acid and Homocysteine Levels in Relation to p53 Expression in Megaloblastic Anemia.

BACKGROUND: Megaloblastic anemia (MBA), also known as macrocytic anemia, is a type of anemia characterized by decreased number of RBCs as well as the presence of unusually large, abnormal and poorly developed erythrocytes (megaloblasts), which fail to enter blood circulation due to their larger size. Lack of vitamin-B12 (VB12) and / or folate (Vitamin-B9, VB9) with elevated homocysteine is the key factor responsible for megaloblastic anemia. Prior studies have demonstrated the induction of apoptosis in these abnormal under-developed erythrocytes. However, it is not clear whether this apoptosis induction is due to elevated p53 level or due to any other mechanism. Furthermore, it is also not fully known whether decreased vitamin-B12 and / or folate are responsible for apoptosis induction mediated by p53 in pre-erythroblasts. METHODS: Levels of serum VB9, VB12 and homocysteine in 50 patients suffering from MBA were compared with 50 non-megaloblastic anemia control subjects, who were referred by the clinicians for bone marrow examination for medical conditions other than MBA. Next, we have measured the p53 expression in the paraffin embedded blocks prepared from bone marrow biopsy, using immunohistochemistry, and the expression levels correlated with VB9 and VB12 levels. RESULTS: Out of 50 MBA patients 40 (80%) and 44 (88%) subjects had very low VB12 and VB9 levels respectively. In contrast, only 2 (4%) and 12 (24%) non-megaloblastic anemia controls, out of 50 subjects, had low VB12 and VB9 respectively. Correlating with low vitamin B9 and B12, the homocysteine levels were high in 80% cases. But, only 20% non-megaloblastic controls exhibited high homocysteine in plasma. Immunohistochemical analysis for p53 expression showed a significantly high level of expression in MBA cases and no-or very low-expression in control subjects. Our correlation studies comparing the VB12 and VB9 levels with p53 expression concludes unusually high p53 levels in patients suffering from VB12 and VB9 deficiency induced MBA compared to control subjects not suffering from MBA. CONCLUSION: Tumor protein p53 is the key protein expressed heavily in the bone marrow biopsies of patients suffering from VB12 and VB9 deficiency induced MBA but not in control subjects. Hence, p53 expression could be used as a surrogate marker for confirming the VB9 and VB12 induced MBA.

Off-therapy procedures are not beneficial in pediatric B-cell acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although curable, approximately 20% of patients relapse. In an effort to detect relapse earlier, our institution performed surveillance bone marrow (BM) and cerebrospinal fluid (CSF) evaluations every 3 months from the end of therapy to 1 year off. This study retrospectively reviewed all patients with B-cell ALL (B-ALL) from September 2005 to September 2010 to determine the benefit and cost of these procedures. Forty-one patients completed therapy and had 190 BMs and 190 lumbar punctures (LPs) performed. Four of 41 patients (9.8%) experienced a relapse. Relapse was detected in only 1 patient by routine BM evaluation (0.5%). Zero LPs were positive. The professional fees for the procedures were $8,738/patient. Therefore, off-therapy BM and CSF evaluations are not effective at detecting relapse and are expensive. Our institution has abandoned off-therapy surveillance for ALL.

Influence of patient and provider characteristics on quality of care for the myelodysplastic syndromes.

Little is known about quality of care for patients with myelodysplastic syndromes (MDS), or patient and provider factors that influence quality. We identified Medicare enrollees diagnosed with MDS between 2006 and 2011, and analysed linked claims for performance on two widely-accepted quality measures: diagnostic bone marrow cytogenetic testing (diagnostic quality) and pre-treatment iron assessment for patients receiving an erythropoiesis-stimulating agent (ESA; treatment quality). A total of 4575 patients met the criteria for diagnostic quality measurement, and 3379 for treatment quality measurement. In the diagnostic cohort, 74% had a claim for marrow cytogenetic testing 3 months before to 3 months after diagnosis. In multivariate models, younger age (P < 0·001), treatment at a higher-volume MDS centre (P < 0·001), and claims for pancytopenia (P < 0·001) were all associated with higher levels of testing. A borderline result was observed for diagnostic year, with improvement over time (P = 0·06). In the treatment cohort, 56% had evidence of pre-ESA iron assessment, with higher rates for later years of diagnosis (P < 0·001), higher household income (P = 0·03), and those treated at higher-volume centres (P = 0·01). In this large cohort of patients with MDS, quality of care was suboptimal overall, but worse in several specific subgroups. These data suggest that targeted educational and/or process-focused interventions are warranted.

Epidemiology, Clinico-Haematological Profile and Management of Aplastic Anaemia: AIIMS Experience.

BACKGROUND: The incidence of aplastic anaemia (AA) is higher in Asia than in the West. The precise incidence of AA in India is not known due to lack of epidemiological study. 20-40% of pancytopenic patients in referral centres are of aplastic anaemia. PATIENTS AND METHODS: This was an analysis of 1501 patients diagnosed with aplastic anaemia over a period of seven and half years (January 2007- June 2014) attending the Aplastic clinic of department of haematology of All India Institute of Medical Sciences, New Delhi. The details regarding medical history, physical examination, complete blood count, bone marrow aspirate and biopsy, treatment received, were retrieved. Inherited bone marrow failure was screened in patients below 35 years. Treatment response was analysed for various treatment modalities. RESULTS: 1501 patients of AA from 20 different states of India were analysed. The bulk of patients were from Uttar Pradesh (28.7%), Bihar (23.6%), Delhi/NCR (20%) and Haryana (7%).The average number of new aplastic anaemia patients enrolled per year 214 (range: 101 -263). The median age at presentation was 25 years (range 2-83),with M;F - 2.3:1. Severity of AA revealed: severe (SAA): 75%, very severe (VSAA): 15%, non-severe (NSAA): 10%. Inherited bone marrow failure syndromes constituted 5% (75 patients) of all aplastic anaemia patients. The most common clinical presentations were pallor (97%), bleeding manifestations (69.6%) and fever (54%). The haematological parameters showed: median level of haemoglobin level: 5.9 gm/dL, WBC: 2700/mm3, ANC: 380/mm3, platelet: 1 0000/mm3. PNH clone was present in 13.5% of patients. 107 patients (7%) were lost to follow up or expired before any treatment was initiated. Only 69 patients (4.5%) received treatment with HLA-matched sibling stem cell transplantation and another 232 (15.5%) patients received ATG plus cyclosporine as immunosuppressive therapy. Seven hundred thirteenpatients (47.5%) received cyclosporine. The overall response to various treatment modalities was: HLA matched sibling haematopoietic stem cell transplant: 75.3%, Anti-thymocyte globulin plus cyclosporine: 58.7%, cyclosporine plus androgen: 45.6%, cyclosporine alone: 32.2%. CONCLUSION: Management of AA is a real challenge in developing countries.This is one of the largest case series from a single centre from India. It is our endeavour to reduce the detrimental outcome by increasing awareness among patients and referring physicians to reduce the delay between diagnosis and treatment.

Diagnostic utility of bone marrow examination for the assessment of patients with fever of unknown origin: a 10-year single-centre experience.

Bone marrow (BM) examination is included in the diagnostic algorithm of fever of unknown origin (FUO), although its role is not clearly determined. The purpose of this study was to assess the role of BM studies in patients with FUO. We retrospectively reviewed 45 consecutive patients (25% human immunodeficiency virus-positive) with FUO who underwent a BM study in the University Hospital of Salamanca from 2000 to 2010. We analysed the diagnostic role of BM smears, multiparameter flow cytometry analysis, histology and microbiological cultures. Five patients (11%) were finally diagnosed by BM study (three had an infectious disease and two were found to have haematological malignancies), all of whom were immunocompetent patients. Histology was the most useful study (diagnosis was obtained in 4/5 patients), while BM cultures did not establish the final diagnosis in any patient. Flow cytometry established the diagnosis in one patient, although this patient was also diagnosed by histology. In conclusion, BM study is useful for establishing the aetiology of FUO. BM biopsy for histological examination should be always mandatory if a BM examination is performed.

Remote and rapid pathological diagnosis in a resource challenged unit.

Malawi is one of the world's poorest countries, but despite this, has a dedicated paediatric oncology service. The service has been hampered by the inability to make a timely cytological diagnosis in the majority of patients. A telemedicine programme was commenced to help overcome this problem, and the results for the first 197 consecutive patients are described. The results are compared with the local reports where available. Most samples were fine needle aspirates (104/197-53%), but others included bone marrow aspirates, peripheral blood films and other fluid collections. A diagnosis was arrived at in 52% of the samples; there were 46 discordant results, 38 were when one or other of the local or distant teams were unable to make a diagnosis, and only 8 where the diagnoses of the 2 teams differed. Diagnoses were made and reports were compiled by the 'distant' team within 24 h and sent to the centre in Malawi. This simple telepathology initiative has had a positive impact on clinical management, and could be used in other less resourced centres twinned with better resourced ones.

[Cardiac tamponade following sternal puncture. Usefulness of ultrasound focussed assessment with sonography for trauma]. / Taponamiento cardíaco tras punción esternal. Utilidad de la ecografía focussed assessment with sonography for trauma.

One of the aims of the medical profession is to be able to detect complications in patients during diagnostic tests and treatments. The early diagnosis of these complications can prevent a fatal outcome. The diagnosis is often based on clinical symptoms and supported by complementary tests. Diagnostic tests have been developed in the last few years that are rapid and easy to use, as well as being cost effective and minimally invasive. Focussed assessment with sonography for trauma ultrasound (echo-FAST) was introduced in the 1990s in the field of resuscitation as a test for the rapid detection of intra-abdominal and pericardial fluid in multiple injury patients, but its uses in other cases not involving trauma still raise doubts and controversy. A case is presented of a patient subjected to a sternal puncture for a bone marrow aspirate, who had a complication of a secondary cardiac tamponade, which was diagnosed early using echo-FAST.