A retrospective study of the incidence and classification of bone marrow disorder in cats (1996–2004)

An 8-year retrospective study of bone marrow reports was conducted to evaluate the incidence and classification of feline bone marrow disorders. Bone marrow reports from 203 cats were reviewed. Blood smears, bone marrow aspiration smears, bone marrow core biopsy specimens, and case records were reviewed for all cats with the exception of those bone marrows reported to be nondiagnostic or cytologically normal. Bone marrows with nonneoplastic/nonmyelodysplastic pathologic changes (n=96) were subclassified into 16 categories. Frequently observed disorders included erythroid hyperplasia, nonregenerative immune-mediated anemia, pure red cell aplasia, aplastic anemia, bone marrow necrosis, myelofibrosis, and lymphocytosis. Dysmyelopoiesis (n=27) was subcategorized into myelodysplastic syndromes (n=20) and secondary (n=7) dysmyelopoiesis. Thirty-four cases of acute leukemia, 3 cases of chronic lymphocytic leukemia, and 14 cases of other neoplasia were identified. These results indicate that a relatively small group of disorders account for the majority of the feline bone marrow disorders. Routine evaluation of blood and bone marrow aspiration smears and core biopsy specimens is needed for proper classification of bone marrow disorders. Ancillary testing, including flow cytometry and immunophenotyping, are useful in some circumstances.These experiments comply with the current laws of the USA     

Bone marrow erythroid precursor Ca++ regulates the response to human recombinant erythropoietin (rHuEPO) in hemodialysis patients

The modulation of erythropoiesis by erythropoietin is conditioned by the concentration of Ca++ in the cytoplasm of the bone marrow erythroid precursors. We evaluated in vitro erythroid colony development from bone marrow erythroid precursors incubated with increasing concentrations of Ca++ or Ca++ plus 1,25 (OH)2 D3, and bone marrow erythroid precursor cytoplasmic Ca++ concentrations in 10 anemic hemodialysis (HD) patients before and during rHuEPO therapy. Results showed that: a) in vitro: in uremics patients before rHuEPO therapy, bone marrow erythroid precursor cytoplasmic Ca++ was lower than in normal subjects; the addition of Ca++ to the bone marrow erythroid precursors induced a dose-dependent Ca++ and erythroid colony development increase; 1,25 (OH)2 D3 potentiated this effect; b) in vivo: rHuEPO normalized bone marrow erythroid precursor Ca++ and erythroid colony development. An inverse correlation was seen between bone marrow erythroid colony development, precursor CA++ before therapy, the in vitro erythroid and the rHuEPO dose needed in vivo to normalize hematological parameters. These data emphasize the role of Ca++ in erythropoiesis and may aid understanding of the mode of action of rHuEPO in HD.     

Defective osteoclast differentiation and function in the osteopetrotic (os) rabbit

We tested the ability of normal osteoclast progenitors found in neonatal liver and bone marrow to develop into functional osteoclasts when co-cultured with metatarsals from newborn osteopetrotic rabbits; the latter inherit an osteoclast incompetence resistant to cure by bone marrow transplantation. This system, developed by Burger and colleagous, has been shown to produce normal, functional osteoclasts when used with normal metatarsals. Our study tested the competence of the mutant skeletal microenvironment for differentiation of normal osteoclasts. Mutant and normal metatarsals were cultured alone or with normal liver, spleen, or bone marrow for up to 14 days. All normal cultures possessed a marrow cavity and contained numerous osteoclasts with cytochemical characteristics (tartrateresistant acid phosphatase) of active cells. Mutant metatarsals co-cultured with normal spleen, liver, or bone marrow failed to develop a marrow cavity (evidence in itself of reduced bone resorption) and had osteoclasts reduced in both numbers and cytochemically detectable activity. Similar metatarsal cultures of an osteopetrotic rat mutation (incisors–absent) curable by bone-marrow transplantation exhibited marrow cavity development in mutant metatarsals co-cultured with normal spleen. These data suggest that the skeletal environment of osteopetrotic rabbits contains an inhibitor or lacks a promoter of osteoclast differentiation and function.     

Marrow uptake index (MUI): a quantitative scintigraphic study of bone marrow in aplastic anaemia

Aplastic anaemia affects the entire bone marrow. Current methods of assessment of bone marrow function, like bone marrow biopsy or peripheral blood examination are either invasive or inadequate and cannot be expected to represent fully the changes in the entire bone marrow tissue. This prospective study was undertaken to develop and standardise a new Nuclear Medicine technique called 'Dynamic Bone marrow Imaging'. Eleven patients and ten controls were studied. Serial images of the pelvis were obtained in frame mode following intravenous injection of 185-370 mBq of 99mTc S. Colloid, and an index, called the Bone Marrow Uptake Index (P) was calculated by taking into consideration the time activity curve obtained over the iliac crest. This was followed by static imaging of the entire bone marrow in all cases. It was possible to obtain excellent information regarding topographic distribution of bone marrow as well as detect early changes in bone marrow function following treatment. An attempt was also made to correlate bone marrow cellularity as obtained by bone marrow biopsy with the results of dynamic bone marrow scintigraphy. On the basis of the encouraging results obtained in the present study, the authors feel that dynamic bone marrow imaging is an excellent technique for the objective evaluation of bone marrow in aplastic anaemia. Aplastic anaemia affects the entire bone marrow tissue. Although much progress has been made in the management of this disease, many aspects of it await better understanding. There is almost total lack of knowledge regarding the distribution of functioning marrow in various phases of aplastic anaemia, such as in relapse and remission. Current methods of assessment of marrow function rely mainly on bone marrow biopsy and peripheral blood examination. Bone marrow biopsy is invasive and cannot be expected to represent fully the changes in the entire tissue. Changes in peripheral blood picture lag behind the changes in the bone marrow. Thus, there is a need for an investigation which is safe, simple, sensitive, non invasive and capable of assessing the global function of bone marrow. Radio-nuclide imaging of bone marrow requires labelling of one or more components of this widely dispersed tissue. The reticuloendothelial and erythropoietic components can be labelled with radio-colloids and radio-iron respectively. Experimental studies have shown that the reticuloendothelial and erythropoietic elements are invariably found together in the marrow and have similar distribution. This report is based on a prospective study of bone marrow function in patients with aplastic anaemia, using 99mTc. Sulphur colloid.     

Argyrophilic nucleolar organiser region (AgNOR) staining in normal bone marrow cells.

Fifteen normal bone marrow aspirates were stained with the agyrophilic nucleolar organiser region (AgNOR) method. The results of the specific staining AgNORs as well as nuclear and cytoplasmic staining were analysed. A system was devised to characterise precisely the AgNORs present in the nuclei of bone marrow cells. Particular types of bone marrow cells had a characteristic AgNOR and non-AgNOR staining pattern. The bone marrow cells were identified easily and reliably with AgNOR staining and the method was especially useful for lymphocytes, plasma cells, erythroid cells, basophils/mast cells, monocytes and cells containing haemosiderin. The immature haemopoietic cells exhibited more and larger AgNORs than the more mature cells. It is concluded that AgNOR staining can be used to study bone marrow cells by providing additional information when used in conjunction with conventional stains.     

Antigenotoxic Effect of Ferulic Acid in 7,12-Dimethyl Benz(a)-Anthracene (DMBA) Induced Genotoxicity

The antigenotoxic effect of ferulic acid was carried out by evaluating the cytogenetic markers, the micronuclei frequency and chromosomal aberrations, in the bone marrow of hamsters in 7,12-dimethylbenz(a)anthracene (DMBA) induced genotoxicity. Genotoxicity was induced in experimental hamsters by single intraperitoneal injection of DMBA (30mg kg⁻¹ b.w). Pretreatment of ferulic acid orally at a dose of 40mg kg⁻¹ b.w for five days significantly reduced the frequency of micronucleated polychromatic erythrocytes (MnPCEs) and the percentage of chromosomal aberrations in hamster''s bone marrow. Our results thus suggest that ferulic acid has potent antigenotoxic effect in DMBA induced genotoxicity in golden Syrian hamsters.     

Novel clonal der(8)t(8;14)(p11;q11),del(9)(q13q22) and t(14;22) (q13;q13) in a patient with fulminant adult T-cell leukemia/lymphoma

We describe a 70-year-old man with fulminant adult T-cell leukemia/lymphoma. He died of progressive disease 1 week after the onset of symptoms. The integrated viral DNA of human T-lymphotropic virus type I was detected in the bone marrow aspirate by polymerase chain reaction. Cytogenetic analysis of bone marrow cells showed novel clonal aberrations consisting of 46,XY,der(8)t(8;14)(p11;q11),del(9) (q13q22),t(14;22)(q13;q13).     

去势大鼠骨髓细胞CD44的表达及其意义

目的：观察CD44在去势大鼠骨髓细胞中的表达情况,探讨CD44对大鼠去势后骨髓细胞分化的作用和意义。方法：采用免疫组织化学方法,观察CD44在SD大鼠去势后4周、8周、12周骨髓细胞中的表达。结果：CD44在去势组和对照组中均有表达,CD44阳性染色主要见于单核细胞融合的多核巨噬细胞／破骨样细胞中,去势后各组动物骨髓细胞CD44表达均显著高于对照组。结论：CD44可能参与大鼠去势后骨髓细胞的分化,可能在骨髓单核细胞向破骨细胞分化中发挥重要作用。     

Bone marrow examination in the koala (Phascolarctos cinereus)

A technique for bone marrow aspiration from the iliac crest in the koala (Phascolarctos cinereus) is described. Bone marrow was obtained from ten healthy koalas under general anaesthesia using the combination of tiletamine HCL and zolazepam HCL. Reference ranges were wide. The mean value for M:E was 1.7 (range 0.8–2.7), the mean percentage proliferating erythroid was 11% (range 5%–16%), and the mean percentage proliferating myeloid was 25% (range 17%–35%). In addition, bone marrow aspiration was performed on 14 koalas with various haematological diseases. Results showed aspiration to be useful in the diagnosis of leukaemia, and the investigation of regenerative anaemia and dysplastic anaemia. It was of varying use in the investigation of non-regenerative anaemia.     

Correction of Fas (CD95) deficiency by haploidentical bone marrow transplantation

Recently, an inherited syndrome characterized by nonmalignant lymphoprolif-eration with autoimmune manifestations, caused by mutations of the Fas (CD95) receptor gene has been described. Because of disease severity, i.e. unremitting lymphoproliferation in a child with complete Fas deficiency, a haploidentical bone marrow transplantation (BMT) was performed despite the known resistance of Fas-deficient lpr mice to bone marrow transplantation. Marrow graft was rejected early; however, a second attempt using bone marrow from the mother led to engraftment and to control of lymphoproliferation and of autoimmune thrombocytopenia up to the last follow-up at 24 months after BMT. This single case shows that resistance to bone marrow engraftment caused by survival of Fas-deficient cells can be overcome.     

Bone marrow examination in the koala (Phascolarctos cinereus)

A technique for bone marrow aspiration from the iliac crest in the koala (Phascolarctos cinereus) is described. Bone marrow was obtained from ten healthy koalas under general anaesthesia using the combination of tiletamine HCL and zolazepam HCL. Reference ranges were wide. The mean value for M:E was 1.7 (range 0.8–2.7), the mean percentage proliferating erythroid was 11% (range 5%–16%), and the mean percentage proliferating myeloid was 25% (range 17%–35%). In addition, bone marrow aspiration was performed on 14 koalas with various haematological diseases. Results showed aspiration to be useful in the diagnosis of leukaemia, and the investigation of regenerative anaemia and dysplastic anaemia. It was of varying use in the investigation of non-regenerative anaemia.     

Abnormal development of B cells and B cell progenitors in autoimmune (NZB x NZW)F1 mice

The (NZB x NZW)F1 (BWF1) autoimmune strain displays reduced numbers of both c mu+ pre-B cells and Ly5(220)+ B cell progenitors in the bone marrow. The loss of these B cell precursor populations in the bone marrow increases with age. In contrast, the bone marrow of BWF1 mice possesses sIg+ B cells comparable in both number and surface phenotype to that observed in conventional strains. Analysis of the surface densities of both sIg and Ly5(220) antigens indicates that BWF1 bone marrow B cells comprise a heterogeneous population of both immature and mature B cells. In addition, BWF1 bone marrow still possessed progenitor cells capable of yielding newly generated B cell precursors and B cells in vitro. The diminished levels of intermediate B cell progenitors observed in BWF1 bone marrow may reflect abnormal regulation of B lineage differentiation during the life span of this autoimmune strain.     

Congenital acute nonlymphoblastic leukemia with translocation (9;18)

Chromosome analysis is becoming an increasingly important tool in the diagnosis and treatment of childhood malignancies. This report illustrates a new translocation t(9;18) in a neonate with congenital acute nonlymphocytic leukemia, which predicted a bone marrow relapse in a normal appearing bone marrow.     

Tetraploidy (92,XXYY) in an acute nonlymphocytic leukemia (M1) patient following autologous bone marrow transplantation

Tetraploidy (4n = 92) without structural chromosome aberrations was found in bone marrow cells of a patient with acute nonlymphocytic leukemia, FAB classification M1, following autologous bone marrow transplantation. This finding, which remained undetected during the remission phases and appeared at relapses, progressed to a hypotetraploid modal number (74 chromosomes) at the terminal phase of the disease. Together with two previous observations in patients with acute lymphoblastic leukemia with L2 morphology and a null cell immunotype, this observation supports the hypothesis that tetraploid karyotypes may represent unusual and aspecific patterns of clonal evolution.     

Myelodysplastisches Syndrom (MDS)

Myelodysplastic syndromes (MDS) are a heterogenous group of clonal stem cell disorders which generally occur in older adults but may also affect children. Primary MDS should be distinguished from secondary MDS associated with antineoplastic or immunosuppressive therapy (t-MDS), exposure to toxic compounds, or genetic disorders. The establishment of a neoplastic clone is reflected by dysplastic features and impaired function which may affect all three hematopoietic cell lineages. The ineffective hematopoiesis which causes bone marrow failure is accompanied by peripheral blood cytopenia and is considered to result from increased apoptosis, at least in the less advanced MDS stages. The elucidation of the molecular pathogenesis of MDS has provided evidence that chromosomal abnormalities are present in about 50% of patients with primary MDS. They include numerical aberrations such as monosomy 5 or 7, trisomy 8, loss of the Y-chromosome and structural abnormalities such as deletion of the long arm of chromosome 5 (5q-syndrome), 7, or 8. Based on the percentage of blasts (<5%, 5-20%, 20-30%) and the presence of >15% ringed sideroblasts for marrows with <5% blasts, the French-American-British (FAB) classifies MDS into 4 morphologic categories: refractory anemia (RA), refractory anemia with excess of blasts (RAEB), refractory anemia with excess of blasts in transformation (RAEB-t), and refractory anemia with ringed sideroblasts. The fifth morphologic type is chronic myelomonocytic leukemia characterized by peripheral blood monocytosis (>1x10(9)/l). However, a modification of this classification will be proposed by the World Health Organization, with the intention of lowering the threshold for the diagnosis of AML from 30% to 20% blast cells. In patients presenting with cytopenias suggesting impaired hematopoiesis, the initial diagnosis depends mainly on the cytological evaluation of bone marrow and blood smears and the histological findings of trephine bone marrow biopsy. In a retrospective analysis we evaluated the occurrence of the distinct FAB-categories as percentage of the total number of MDS cases diagnosed at the Institute of Pathology of the University of Freiburg. A total of 63% fullfilled the criteria of RA/RARS, 17% of RAEB, 14% of RAEB-t, and 6% of CMML. A fibrotic variant of MDS was observed in 7.67% of all cases, ranging from 2.34% in RA up to 15. 42-15.84% in the categories which did not show significant differences with regard to myelofibrosis. The histologic evaluation of a trephine bone marrow biopsy is of critical importance for the evaluation of fibrotic or hypocellular MDS since these patterns are not reflected by the cytological examination. The combined cytological and histological diagnosis of bone marrow and peripheral blood is a reliable tool for the initial diagnosis of MDS. In addition, cytogenetic and molecular analysis should be performed. Presently, the risk of leukemic transformation is evaluated using the International Prognostic Scoring System for MDS, which is the sum of the scores of bone marrow blasts, karyotypes and cytopenia. In the context of clinical trials therapeutic modalities should be considerd according to the age and the general performance state and the prognostic scores of individual patients.     

Impaired Ca2+ mobilization by X-linked agammaglobulinaemia (XLA) B cells in response to ligation of the B cell receptor (BCR)

XLA bone marrow samples were shown to contain B cells expressing IgM, and pre-B cells that express the μ-surrogate light chain (μψLC) complex, albeit at a reduced frequency to that found in normal bone marrow. Antibody ligation of μ heavy chain on these cells and an XLA B cell line did not induce a Ca²⁺ flux, whereas ligation of μ heavy chain on normal bone marrow cells, μψLC⁺ pre-B cell lines and an IgM⁺ B cell line did. The block in XLA B cells was not due to a defect in the basic mechanism of Ca²⁺ flux generation, as the cells responded well to thapsigargin. In addition, the defect did not affect T cells, which were shown to respond to CD3 antibody with a Ca²⁺ flux. Ligation of μ heavy chain on XLA bone marrow cells did, however, activate tyrosine kinases, resulting in tyrosine phosphorylation of a cellular protein with a molecular weight of approximately 115 kD. These results indicate that Btk may be necessary for the generation of the Ca²⁺ flux in response to ligation of μ heavy chain on B cells and μψLC⁺ pre-B.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity

The selective toxicity of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) for the thymus, consisting primarily of immature T-cells, led us to search for an analogous selective toxicity for the immature B-lymphocytes in the bone marrow. In the dose-response study C57B1/6 male mice were injected with either vehicle alone (corn oil), 30, 60, or 120 micrograms/kg of TCDD i.p. The mice were killed by cervical dislocation 7 days later. In the time-response study, mice were injected with either saline or 120 micrograms/kg i.p. TCDD, 3, 7, 14, or 21 days before killing. In both studies, the following were analyzed: change in body weight, thymus weight, spleen and bone marrow cellularity, and spleen and marrow B-lymphocyte function, measured using the in vitro B-lymphocyte colony forming unit in culture assay, with the mitogen lipopolysaccharide (LPS) from Salmonella typhosa, and the in vitro plaque forming cell assay, with the thymus independent antigen, TNP-LPS. In the dose-response study there was a reduction in thymic weight, spleen B-cell functional response (per spleen), and bone marrow B-cell functional response to 14%, 35-54%, and 20-32% of control, respectively, at a dosage of 120 micrograms/kg. In the time-response study, thymic weight and bone marrow B-cell functional response (per femur) were reduced to 6% and 18% of control, respectively, at day 21. The results indicate that TCDD was selectively more toxic to the immature B-cells in the bone marrow than the more mature B-cells in the spleen. This immunotoxicity was dose-dependent.     

Unilateral radial aplasia and trisomy 22 mosaicism.

A child with unilateral radial aplasia, asymmetry, other malformations, and severe physical and mental retardation is reported. In blood and bone marrow cultures a low mosaicism for trisomy 22 was found. In a few cells a chromosome 22 was missing. The importance of early cytogenetic analysis on large numbers of cells is emphasised, especially in cases of asymmetry where mosaicism is suspected.     

Treatment of stroke with (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate and bone marrow stromal cells upregulates angiopoietin-1/Tie2 and enhances neovascularization

Neovascularization may contribute to functional recovery after neural injury. Combination treatment of stroke with a nitric oxide donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETA-NONOate) and bone marrow stromal cells promotes functional recovery. However, the mechanisms underlying functional improvement have not been elucidated. In this study, we tested the hypothesis that combination treatment upregulates angiopoietin-1 and its receptor Tie2 in the ischemic brain and bone marrow stromal cells, thereby enhancing cerebral neovascularization after stroke. Adult wild type male C57BL/6 mice were i.v. administered PBS, bone marrow stromal cells 5x10(5), DETA-NONOate 0.4 mg/kg or combination DETA-NONOate with bone marrow stromal cells (n=12/group) after middle cerebral artery occlusion. Combination treatment significantly upregulated angiopoietin-1/Tie2 and tight junction protein (occludin) expression, and increased the number, diameter and perimeter of blood vessels in the ischemic brain compared with vehicle control (mean+/-S.E., P<0.05). In vitro, DETA-NONOate significantly increased angiopoietin-1/Tie2 protein (n=6/group) and Tie2 mRNA (n=3/group) expression in bone marrow stromal cells. DETA-NONOate also significantly increased angiopoietin-1 protein (n=6/group) and mRNA (n=3/group) expression in mouse brain endothelial cells (P<0.05). Angiopoietin-1 mRNA (n=3/group) was significantly increased in mouse brain endothelial cells treated with DETA-NONOate in combination with bone marrow stromal cell-conditioned medium compared with cells treated with bone marrow stromal cell-conditioned medium or DETA-NONOate alone. Mouse brain endothelial cell capillary tube-like formation assays (n=6/group) showed that angiopoietin-1 peptide, the supernatant of bone marrow stromal cells and DETA-NONOate significantly increased capillary tube formation compared with vehicle control. Combination treatment significantly increased capillary tube formation compared with DETA-NONOate treatment alone. Inhibition of angiopoietin-1 significantly attenuated combination treatment-induced tube formation. Our data indicated that combination treatment of stroke with DETA-NONOate and bone marrow stromal cells promotes neovascularization, which is at least partially mediated by upregulation of the angiopoietin-1/Tie2 axis.     

Effects of Anti-T Cell (Theta) and Anti-B Cell (Beta) Serum on the Immune Response in Mice

Heterologous anti-B cell (anti-beta) serum was prepared in rabbits against the spleen from neonatally thymectomized mice. The anti-beta serum, after absorption with thymus, is cytotoxic for bone marrow, bone marrow-derived cells, fetal liver and peritoneal lymphocytes. The cytotoxicity to the B cell can be absorbed out with bone marrow.

The cytotoxic effects of anti-beta serum on spleen and lymph node cells is compared to that of anti-theta serum. The data suggest that spleen has relatively more B than T cells, while lymph node has relatively more theta-positive cells.

To test the effect of anti-beta and anti-theta serum on the functional activity of lymphoid cells, C57 spleen or thymus was pre-incubated with the antiserum, in the presence of complement, and tested in vivo for graft-vs-host activity or transfer of an adoptive immune response to SRBC.

Treatment with anti-beta serum does not decrease the graft-vs-host activity of thymus or spleen cells. Anti-theta serum does decrease the graft-vs-host activity of both thymus and spleen cells.

Neither anti-beta serum nor anti-theta serum affect the phagocytic activity of peritoneal macrophages.

Both anti-beta serum and anti-theta serum decrease the transfer of an adoptive primary and secondary immune response to SRBC. A combination of anti-theta and anti-beta treated spleen can transfer adoptive immunity. Thymus and bone marrow can reconstitute the immunocompetence of anti-theta or anti-beta treated spleen respectively.

The results suggest that T cells alone can mount a graft-vs-host reaction and that this activity is not affected by anti-beta serum. The transfer of a humoral antibody response, on the other hand, requires functionally active T- and B-cells. This holds true for a primary as well as secondary immune response. Our anti-beta serum does not appear to have any anti-macrophage activity.