Haematological profiles after Intensive phase of Anti Koch Treatment with special emphasis on bone marrow changes.

BACKGROUND: Tuberculosis remains a major public health problem in various parts of the world. It leads to various haematological changes. Study of these haematological changes will help better patient management. OBJECTIVE & METHODS: It is to evaluate haematological changes in tuberculosis patients and compare the result with special emphasis to bone marrow changes as active case search is sharply decreasing the miliary tuberculosis. It is also to evaluate the patients with before and after the Intensive Phase of Anti Koch Treatment. Sputum positive and sputum negative tuberculosis patients confirmed by other ancillary techniques were included into this study. It is conducted at a tertiary level hospital in rural area. RESULT: In this study bone marrow hypercellularity was of erythroid series with only 1.92% patients showed granuloma in bone marrow aspiration. In addition to bone marrow changes, significant changes were evident in haemoglobin level, Erythrocyte Sedimentation Rate (ESR) Total White Blood Cell count and RBC count. DISCUSSION: In majority cases this study showed Erythroid Hyperplasia. It is sharp contrast with other study where myeloid hyperplasia was evident. This study also differs from other study where high number of bone marrow granuloma was reported. In this study only 1.92% cases showed bone marrow granuloma. This study also documented higher number of anaemic cases mostly because of the institute serves poor and tribal population. CONCLUSION: In our study the cases showing granuloma and hyperplasia of myeloid series were limited. With introduction of Directly Observed Treatment and house to house active case search helped to sharply decrease bone marrow granuloma by limiting multi-organ spread. This study showed, ESR level may be considered as prognostic parameters of tuberculosis.

Bone Marrow Examination: Why, How, and What to Expect from the Pathologist.

This article describes the indications for sampling of bone marrow, the technical aspects of obtaining marrow core biopsies and aspirates, and the preparation of marrow smears. All aspects are illustrated with clinical cases. The information that can be expected from the pathologist's report of marrow samples is outlined, and the clinical features and prognosis of different types of leukemia are detailed.

Nonproliferative and Proliferative Lesions of the Rat and Mouse Hematolymphoid System.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative changes in rats and mice. The purpose of this publication is to provide a standardized nomenclature for classifying changes observed in the hematolymphoid organs, including the bone marrow, thymus, spleen, lymph nodes, mucosa-associated lymphoid tissues, and other lymphoid tissues (serosa-associated lymphoid clusters and tertiary lymphoid structures) with color photomicrographs illustrating examples of the lesions. Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. The nomenclature for these organs is divided into 3 terminologies: descriptive, conventional, and enhanced. Three terms are listed for each diagnosis. The rationale for this approach and guidance for its application to toxicologic pathology are described in detail below.

Discovery of a bone-like blood particle in the peripheral circulation of humans and rodents.

OBJECTIVE: To characterize ossified bone marrow blood vessels and confirm the presence of ossified particles (OSP) in humans and rodents. METHODS: Human bone marrow blood vessels were processed for scanning and transmission electron microscopy. Whole blood samples were collected from younger (26-39 years; n = 6) and older (55-63 years; n = 6) volunteers and male Fischer-344 rats (1 month, n = 7; 6 months, n = 7; 12 months, n = 7; 18-months, n = 6; 24 months, n = 8). OSP in the whole blood samples were sorted and imaged with microscopy to determine diameter, circularity, and solidity. Additionally, the chemical composition of OSP was determined via elemental analysis. RESULTS: SEM revealed two types of ossified bone marrow blood vessels: that is, "transitioning" and "ossified." OSP were adhered to the surface of transitioning vessels and theoretically gain access to and circulate within the blood. The majority of OSP were ≤15 µm in diameter, but many were of sufficient size to serve as emboli (ie, >15 µm).OSP were predominately oblong in shape and several had jagged tips and edges. CONCLUSIONS: We introduce a novel, bone-like blood particle that may be diagnostic of bone marrow blood vessel ossification. Further, OSP may associate with several disease states (eg, atherosclerosis).

A Case of Acquired Amegakaryocytic Thrombocytopenia with Anti-c-mpl Autoantibody: Comparison with Idiopathic Thrombocytopenic Purpura.

Acquired amegakaryocytic thrombocytopenia (AAMT) is a rare disease that causes severe bleeding. The pathogenesis and treatment of AAMT have not yet been defined. We report the case of a 60-year-old woman diagnosed with AAMT, who presented with severe thrombocytopenia, gastroin-testinal bleeding, and significantly reduced bone marrow megakaryocytes. The patient was treated with methylprednisolone, cyclosporin, and intravenous immunoglobulin. After 2 weeks of treatment, her platelet count started to increase, and her bone marrow megakaryocyte count had normalized 3 months after diagnosis. At the time of diagnosis, the patient was seropositive for anti-c-mpl antibody but was seen to be seronegative once the platelet count recovered. In contrast, anti-c-mpl antibodies were not detected in the serum of 3 patients with idiopathic thrombocytopenic purpura. This case study suggests that anti-c-mpl antibody plays an important role in the development of AAMT, and that intensive immunosuppressive treatment is required for autoantibody clearance and recovery of megakaryocyte count.

Peripheral blood immunophenotyping in a large cohort of patients with Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is one of the more common inherited bone marrow failure syndromes, characterized by neutropenia, occasional thrombocytopenia, and anemia. Bone marrow evaluation reveals an increased number of monocytes and mature B cells along with decreased granulocytes. However, little is known about the subpopulations of peripheral blood cells, and few previous publications have been based on a small number of patients. Here, we report a comprehensive immunophenotypic analysis from a cohort of 37 SDS patients who display impairment mostly in the myeloid compartment with a deficiency also in the number of B cells and CD4/CD8 double-negative T cells.

Macropolycyte in Pediatrics.

Macropolycytes are tetraploid neutrophils produced during accelerated myelopoiesis. They have been reported in adults with pernicious anemia, sepsis, and after cytotoxic chemotherapy. Two pediatric cases are reported, one after granulocyte colony-stimulating factor treatment and the other following Kawasaki disease, respectively.

Increased serum levels of MIC1/GDF15 correlated with bone erosion in spondyloarthritis: A pilot study.

INTRODUCTION: To assess the association between growth differentiation factor-15 (GDF15) and radiographic features including bone marrow edema and bone erosion in Spondyloarthritis (SpA). METHODS: Patients with SpA (n = 120) receiving treatment in the Guangdong General Hospital, China, between August 2012 and December 2016 were retrospectively included. Serum of patients and healthy controls (n = 30) were collected and GDF15 levels were measured using ELISA. Inflammation was assessed by C-reactive protein (CRP), and magnetic resonance imaging (MRI) of the sacroiliac joint using Spondyloarthritis Research Consortium of Canada score and a method of dichotomy to assess fat metaplasia, bone erosion, and ankylosis. Radiographs of the pelvis were scored using the modified New York (mNY) score. RESULTS: Serum GDF15 levels were higher in SpA patients compared to controls (503.52 ±â€Š222.92 vs. 190.86 ±â€Š104.18 pg/mL, P < .0001). Patients who suffered from bone erosion on MRI had higher levels of GDF15 (525.72 [186.33, 801.62]vs. 428.06 [255.15, 670.98] pg/mL, P = .0375). There was a positive correlation between serum GDF15 and CRP (r = 0.5442, P < .0001). Moreover, GDF15 levels were related to CRP levels (r = 0.5658, P < .0001) in those X-ray scores were III, according to 1984mNY criteria. Receiver operating characteristic (ROC) analysis showed that GDF15 levels above 501.98pg/mL could predict presence of bone erosion on MRI. CONCLUSION: The present study suggested that serum GDF15 levels are higher in SpA patients than in healthy controls. The GDF15 level was correlated with CRP and may be a surrogate biomarker in bone erosion.

Establishment of a flow cytometry assay for detecting paroxysmal nocturnal hemoglobinuria-type cells specific to patients with bone marrow failure.

Minor populations of glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells in the peripheral blood may have a prognostic value in bone marrow failure (BMF). Our objective is to establish the optimal flow cytometry (FCM) assay that can discriminate GPI(-) populations specific to BMF from those of healthy individuals. To identify a cut-off that discriminates GPI(-) rare cells from GPI(+) cells, we determined a position of the borderline that separates the GPI(-) from GPI(+) cells on a scattergram by testing more than 30 healthy individuals, such that no GPI(-) dot fell into the upper left quadrant where fluorescein-labeled aerolysin (FLAER)-CD11b+ granulocytes and CD55-CD59- glycophorin A+ erythrocytes were positioned. This method allowed us to define ≥ 0.003% CD11b+FLAER- granulocytes and ≥ 0.005% glycophorin A+CD55-CD59- erythrocytes to be specific to BMF patients. Longitudinal cross-validation studies showed minimal (< 0.02%) inter-laboratory differences in the GPI(-) cell percentage. An analysis of 1210 patients with BMF revealed a GPI(-) cell population in 56.3% of patients with aplastic anemia and 18.5% of patients with myelodysplastic syndrome. The GPI(-) granulocyte percentages was 0.003-0.01% in 3.7% of patients. This FCM assay effectively identified an increase in the percentage of GPI(-) rare cells that are specific to BMF patients and allowed different laboratories to accurately detect 0.003-0.01% of pathological GPI(-) cells.

Utility of the immature platelet fraction in pediatric immune thrombocytopenia: Differentiating from bone marrow failure and predicting bleeding risk.

BACKGROUND: Differentiating childhood immune thrombocytopenia (ITP) from other cause of thrombocytopenia remains a diagnosis of exclusion. Additionally factors that predict bleeding risk for those patients with ITP are currently not well understood. Previous small studies have suggested that immature platelet fraction (IPF) may differentiate ITP from other causes of thrombocytopenia and in combination with other factors may predict bleeding risk. METHODS: We performed a retrospective chart review of thrombocytopenic patients with an IPF measured between November 1, 2013 and July 1, 2015. Patients were between 2 months and 21 years of age with a platelet count <50 × 109 /l. Each patient chart was reviewed for final diagnosis and bleeding symptoms. A bleeding severity score was retrospectively assigned. RESULTS: Two hundred seventy two patients met inclusion criteria, 97 with ITP, 11 with bone marrow failure (BMF), 126 with malignancy, and 38 with other causes of thrombocytopenia. An IPF > 5.2% differentiated ITP from BMF with 93% sensitivity and 91% specificity. Absolute immature platelet number (AIPN) was significantly lower in ITP patients with severe to life-threatening hemorrhage than those without, despite similar platelet counts. On multivariate analysis, an IPF < 10.4% was confirmed as an independent predictor of bleeding risk at platelet counts <10 × 109 /l in patients with ITP. CONCLUSIONS: IPF measurement alone has utility in both the diagnosis of ITP and identifying patients at increased risk of hemorrhage. Further study is required to understand the pathophysiological differences of ITP patients with lower IPF/AIPN.

Acquired amegakaryocytic thrombocytopenia previously diagnosed as idiopathic thrombocytopenic purpura in a patient with hepatitis C virus infection.

We report the first case of a patient with hepatitis C virus (HCV) infection and idiopathic thrombocytopenic purpura (ITP), who later developed acquired amegakaryocytic thrombocytopenia (AAMT), with autoantibodies to the thrombopoietin (TPO) receptor (c-Mpl). A 64-year-old woman, with chronic hepatitis C, developed severe thrombocytopenia and was diagnosed with ITP. She died of liver failure. Autopsy revealed cirrhosis and liver carcinoma. In the bone marrow, a marked reduction in the number of megakaryocytes was observed, while other cell lineages were preserved. Therefore, she was diagnosed with AAMT. Additionally, autoantibodies to c-Mpl were detected in her serum. Autoantibodies to c-Mpl are one of the causes of AAMT, acting through inhibition of TPO function, megakaryocytic maturation, and platelet formation. HCV infection induces several autoantibodies. HCV infection might also induce autoantibodies to c-Mpl, resulting in the development of AAMT. This mechanism may be one of the causes of thrombocytopenia in patients with HCV infection.

p53 Mediates Failure of Human Definitive Hematopoiesis in Dyskeratosis Congenita.

Dyskeratosis congenita (DC) is a bone marrow failure syndrome associated with telomere dysfunction. The progression and molecular determinants of hematopoietic failure in DC remain poorly understood. Here, we use the directed differentiation of human embryonic stem cells harboring clinically relevant mutations in telomerase to understand the consequences of DC-associated mutations on the primitive and definitive hematopoietic programs. Interestingly, telomere shortening does not broadly impair hematopoiesis, as primitive hematopoiesis is not impaired in DC cells. In contrast, while phenotypic definitive hemogenic endothelium is specified, the endothelial-to-hematopoietic transition is impaired in cells with shortened telomeres. This failure is caused by DNA damage accrual and is mediated by p53 stabilization. These observations indicate that detrimental effects of telomere shortening in the hematopoietic system are specific to the definitive hematopoietic lineages. This work illustrates how telomere dysfunction impairs hematopoietic development and creates a robust platform for therapeutic discovery for treatment of DC patients.

Long-term systolic function in children and young adults after hematopoietic stem cell transplant.

Congestive heart failure and subclinical left ventricular systolic dysfunction (LVSD) affect long-term survivors of hematopoietic stem cell transplant (HSCT). Echocardiographic measurements of global longitudinal and circumferential strain have shown promise in identifying subclinical LVSD in cancer survivors. We analyzed echocardiograms in 95 children and young adults with malignancies or bone marrow failure syndromes performed before HSCT and 1-6 years after HSCT. We additionally measured the biomarkers soluble suppression of tumorigenicity-2 (sST-2) and cardiac troponin-I (cTn-I) in the same children through 49 days post HSCT. Ejection fraction (EF) after HSCT was unchanged from baseline (baseline: z-score -0.73 vs long-term follow up: -0.44, P=0.11). Global longitudinal strain was unchanged from baseline (-20.66 vs -20.74%, P=0.90) as was global circumferential strain (-24.3 vs -23.5%, P=0.32). Levels of sST-2 were elevated at all time points compared with baseline samples and cTn-I was elevated at days 14 and 28. Cardiac biomarkers at any time point did not correlate with long-term follow-up EF. In children and young adult survivors of HSCT, EF was unchanged in the first years after HSCT. Elevation in cardiac biomarkers occurring after HSCT suggest subclinical cardiac injury occurs in many patients and long-term monitoring for LVSD should continue.

The effect of variation in donor platelet function on transfusion outcome: a semirandomized controlled trial.

The effect of variation in platelet function in platelet donors on patient outcome following platelet transfusion is unknown. This trial assessed the hypothesis that platelets collected from donors with highly responsive platelets to agonists in vitro assessed by flow cytometry (high-responder donors) are cleared more quickly from the circulation than those from low-responder donors, resulting in lower platelet count increments following transfusion. This parallel group, semirandomized double-blinded trial was conducted in a single center in the United Kingdom. Eligible patients were those 16 or older with thrombocytopenia secondary to bone marrow failure, requiring prophylactic platelet transfusion. Patients were randomly assigned to receive a platelet donation from a high- or low-responder donor when both were available, or when only 1 type of platelet was available, patients received that. Participants, investigators, and those assessing outcomes were masked to group assignment. The primary end point was the platelet count increment 10 to 90 minutes following transfusion. Analysis was by intention to treat. Fifty-one patients were assigned to receive platelets from low-responder donors, and 49 from high-responder donors (47 of which were randomized and 53 nonrandomized). There was no significant difference in platelet count increment 10 to 90 minutes following transfusion in patients receiving platelets from high-responder (mean, 21.0 × 109/L; 95% confidence interval [CI], 4.9-37.2) or low-responder (mean, 23.3 × 109/L; 95% CI, 7.8-38.9) donors (mean difference, 2.3; 95% CI, -1.1 to 5.7; P = .18). These results support the current policy of not selecting platelet donors on the basis of platelet function for prophylactic platelet transfusion.

A retrospective study of paroxysmal nocturnal hemoglobinuria in pediatric and adolescent patients.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease, especially in children, characterized by intravascular hemolysis, thrombotic events, serious infections and bone marrow failure. We describe 16 patients who were diagnosed with PNH in childhood or adolescence. The time interval between the onset of symptoms and the PNH diagnosis and its treatment were compared in patients with classic PNH versus PNH associated with bone marrow disorder (PNH/BMD). A greater delay in diagnosis was observed in classic PNH compared to PNH/BMD patients. The first group of patients had higher levels of LDH, total bilirubin and absolute reticulocyte count and a bigger PNH clone size compared to PNH/BMD patients; also thrombotic events were observed only in the classic form of PNH. Conversely, PNH/BMD patients showed lower median levels of platelets. Apart from standard supportive measures, four patients with classic PNH received eculizumab whereas four patients with PNH/BMD underwent hematopoietic stem cell transplantation. Our series confirm that the most frequent presentation of PNH in the pediatric-adolescent age is PNH/BMD. The delay between the onset of symptoms and PNH diagnosis is relevant principally in the classic form. Moreover, our study showed that any case of unexpected thrombosis represents a criterium to perform a PNH screening.

Denosumab is effective in the treatment of bone marrow oedema syndrome.

Bone marrow oedema (BMO) syndrome describes a painful condition with increase of interstitial fluid within bone and is often lately diagnosed due to unspecific symptoms. The underlying causes are diverse while it is widely assumed that in cases of BMO local bone resorption is increased. Denosumab, a human monoclonal antibody that binds to the receptor activator of nuclear factor kappa-B ligand (RANKL) inhibits osteoclastic bone resorption and is commonly administered in the treatment of osteoporosis. Besides one previous case report, its clinical effectiveness in the treatment of bone marrow oedema has not been elucidated. We treated 14 patients with primary (idiopathic) bone marrow oedema of the lower extremity with single dose denosumab application. Mean time between onset of pain and therapy was 155days. MRI scans were performed for initial diagnosis, and 6-12 weeks after denosumab injection. Vitamin D and calcium homeostasis were strived to be balanced before initiation of therapy. Furthermore bone status was analysed using Dual-energy X-ray absorptiometry (DXA) and extended bone turnover serum markers. After 6-12 weeks, BMO dissolved partly or completely in 93%, while a complete recovery was observed in 50% of the individuals. Visual analogue scale (VAS) evaluation revealed a significant decrease in pain level. Furthermore, bone turnover decreased significantly after treatment. No adverse reactions were reported. In conclusion, our retrospective analysis shows that denosumab is highly effective in the treatment of bone marrow oedema and therefore represents an alternative treatment option.

Bone Marrow Graft-Versus-Host Disease in Major Histocompatibility Complex-Matched Murine Reduced-Intensity Allogeneic Hemopoietic Cell Transplantation.

BACKGROUND: Most clinical allogeneic hemopoietic cell transplants (alloHCT) are now performed using reduced-intensity conditioning (RIC) instead of myeloablative conditioning (MAC); however, the biology underlying this treatment remains incompletely understood. METHODS: We investigated a murine model of major histocompatibility complex-matched multiple minor histocompatibility antigen-mismatched alloHCT using bone marrow (BM) cells and splenocytes from B6 (H-2) donor mice transplanted into BALB.B (H-2) recipients after RIC with fludarabine of 100 mg/kg per day for 5 days, cyclophosphamide of 60 mg/kg per day for 2 days, and total body irradiation (TBI). RESULTS: The lowest TBI dose capable of achieving complete donor chimerism in this mouse strain combination was 325 cGy given as a single fraction. Mice that underwent RIC had a reduced incidence and delayed onset of graft-versus-host disease (GVHD) and significantly prolonged survival compared with MAC-transplanted recipients (TBI of 850 cGy plus cyclophosphamide of 60 mg/kg per day for 2 days). Compared with syngeneic controls, RIC mice with GVHD showed evidence of BM suppression, have anemia, reduced BM cellularity, and showed profound reduction in BM B cell lymphopoiesis associated with damage to the endosteal BM niche. This was associated with an increase in BM CD8 effector T cells in RIC mice and elevated blood and BM plasma levels of T helper1 cytokines. Increasing doses of splenocytes resulted in increased incidence of GVHD in RIC mice. CONCLUSIONS: We demonstrate that the BM is a major target organ of GVHD in an informative clinically relevant RIC mouse major histocompatibility complex-matched alloHCT model by a process that seems to be driven by CD8 effector T cells.

Value of CD16/CD66b/CD45 in comparison to CD55/CD59/CD45 in diagnosis of paroxysmal nocturnal haemoglobinuria: An Indian experience.

BACKGROUND & OBJECTIVES: Diagnosis of paroxysmal nocturnal haemoglobinuria (PNH), a rare haematopoietic stem cell disorder, is challenging in patients with bone marrow failure (BMF) syndrome like aplastic anaemia (AA). This study was conducted with the aim to test the efficacy of the newly recommended markers viz. anti-CD16 and CD66b antibody over the existing anti-CD55 and CD59 antibody for PNH diagnosis in India. METHODS: This study was conducted on 193 suspected cases of PNH by flow cytometry using lyse wash technique to stain the granulocytes with CD16/CD66b and CD55/CD59. RESULTS: Of the 193 suspected cases, 62 patients showed the presence of PNH clone. Forty six patients were detected by CD55/CD59/CD45, whereas 61 were detected by CD16/CD66b/CD45. CD16/CD66b detected 16 (25.8%) additional patients over CD55/CD59 (P<0.05) and was more sensitive in detecting the PNH clone with higher negative predictive value. Most of the patients (11/16) who were picked up by CD16/CD66b were of AA who had small clone sizes. Further, the PNH clones were more discreetly identified in CD16/CD66b plots than by CD55/CD59. Clone size assessed by CD16/CD66b which reflects the clinical severity of classical PNH (thrombosis/haemolysis), was more representative of the underlying clinical condition than CD55/59. INTERPRETATION & CONCLUSIONS: In our experience of 62 patients of PNH, CD16/CD66b proved to be more efficacious in detecting PNH. The new panel was especially useful in monitoring PNH associated with BMF which had small clone sizes.

Imaging of Bone Marrow.

Bone marrow is the essential for function of hematopoiesis, which is vital for the normal functioning of the body. Bone marrow disorders or dysfunctions may be evaluated by blood workup, peripheral smears, marrow biopsy, plain radiographs, computed tomography (CT), MRI and nuclear medicine scan. It is important to distinguish normal spinal marrow from pathology to avoid missing a pathology or misinterpreting normal changes, either of which may result in further testing and increased health care costs. This article focuses on the diffuse bone marrow pathologies, because the majority of the bone marrow pathologies related to hematologic disorders are diffuse.