Association of alkaline-phosphatase-positive reticulum cells in bone marrow with granulocytic precursors

In the bone marrow, an elaborate stroma forms the structural basis of the hemopoietic microenvironment. In this study, two different types of stromal cells were identified with certainty on tissue sections of intact bone marrow of rats and mice using light and electron microscopic histochemistry: (a) a fibroblast-type of reticulum cell which is characterized by having alkaline phosphatase associated with its plasma membrane. We refer to this cell as the alkaline-phosphatase- positive reticulum cell (Al-RC). It is closely associated with granulocytic precursors, particularly myeloblasts and neutrophilic promyelocytes. These reticulum cells may be found throughout the marrow but are concentrated near the endosteum. (b) a macrophage-type of reticulum cell which is characterized by its abundance of lysosomal acid phosphatase and is mainly associated with erythroid precursors (as observed by others). In contrast to the above-mentioned cell type, this latter cell was found to be distributed uniformly throughout the marrow. We speculate that the Al-RC are mesenchymal stromal cells necessary for granulocytic differentiation in bone marrow.     

Marrow erythroid and neutrophil cellularity in the dog.

This paper describes a method for determining the number of marrow erythroid and neutrophil cells in which the cellularity of marrow sections was related to that of the total marrow by radioiron dilution. Tissue sections were prepared from methacrylate-embedded dog marrow biopsies, and neutrophils were identified by staining of their primary granules. After correction of direct section counts for multiple counting error, accurate neutrophil-erythroid ratios were established with a coefficient of variation of less than 10 percent when 10-4 cells were examined. An average neutrophil-erythroid ratio of 1.2 was found in six normal dogs. The total number of nucleated red cells in the dog was 5.48 plus or minus 0.78 times 10-9/kg (plus or minus 1 SD), and the corresponding erythron iron turnover was 0.90 plus or minus 0.11 mg Fe/100 ml whole blood/day. The total number of marrow neutrophils, derived from the neutrophil-erythroid ratio, was 6.6 plus or minus 0.59 times 10-9 cells/kg, of which 1.4 were promyelocytes and myelocytes, 2.3 were metamyelocytes and bands, and 3.0 were segmented neutrophils. Leukopheresis studies were carried out in six dogs to confirm the accuracy of these cellular measurements. Marrow counts showed a mean decrease of 22.7 times 10-9 cells or 35 percent of the postmitotic neutrophil pool, and it was calculated that 10.2 times 10-9 additional cells had been taken from already circulating blood. This estimated deficit of 32.9 times 10-9 was almost identical to the 33 times 10-9 cells actually counted in the removed blood.     

Neutrophil kinetics in man.

A method has been developed for measuring neutrophil cellularity in normal human bone marrow, in which the neutrophil-erythroid ratio was determined from marrow sections and marrow normoblasts were estimated by the erythron iron turnover. Neutrophil maturational categories, defined by morphologic criteria, were supported by autoradiographs of marrow flashed-labeled with 3H-thymidine. Correction for multiple counting error was empirically derived by counting serial sections through cells of each maturational category. The normal neutrophil-erythroid ratio in 13 normal human subjects was 1.5 +/- 0.07. The mean number of normoblasts in the same subjects was estimated to be 5.07 +/- 0.84 X 10(9) cells/kg. Total marrow neutrophils (X 10(9) cells/kg) were 7.70 +/- 1.20, the postmitotic pool (metamyelocytes, bands, and segmented forms) was 5.59 +/- 0.90 and the mitotic pool (promyelocytes + myelocytes) was 2.11 +/- 0.36. Marrow neutrophil ("total") production has been determined from the number of neutrophils comprising the postmitotic marrow pool divided by their transit time Transit time was derived from the appearance in circulating neutrophils of injected 3H-thymidine. The postmitotic pool comprised 5.59 +/- 0.90 X 10(9) neutrophils/kg, and the transit time was 6.60 +/- 0.03 days. From these data marrow neutrophil production was calculated to be 0.85 X 10(9) cells/kg per day. Effective production, measured as the turnover of circulating neutrophils labeled with 3H-thymidine, was 0.87 +/- 0.13 X 10(9) cells/kg per day. This value correlated well with the calculation of marrow neutrophil production. A larger turnover of 1.62 +/- 0.46 X 10(9) cells/kg per day was obtained when diisopropylfluorophosphate-32P was used to label circulating neutrophils. Studies using isologous cells doubly labeled with 3H-thymidine and diisopropylfluorophosphate-32P demonstrated a lower recovery and shorter t1/2 of the 32P label.     

Committed T lymphocyte stem cells of rats. Characterization by surface W3/13 antigen and radiosensitivity

The existence of stem cells committed to the T lymphoid lineage was deduced from studying how rat T and B stem cells differ in their expression of membrane W3/13 antigen and in their susceptibility in vivo to gamma irradiation. Stem cell activity of rat bone marrow and fetal liver was measured in long-term radiation chimeras using B and T cell alloantigenic surface markers to identify the progeny of donor cells. Monoclonal mouse anti-rat thymocyte antibody W3/13 labeled approximately 40% of fetal liver cells and 60-70% of young rat bone marrow cells (40% brightly, 25% dimly). Bright, dim, and negative cells were separated on a fluorescence-activated cell sorter. All B and T lymphoid stem cells in fetal liver were W3/13 bright, as were B lymphoid stem cells in bone marrow. W3/13 dim bone marrow had over half the T cell repopulating activity of unseparated marrow but gave virtually no B cell repopulation. In further experiments, the radiosensitivity of endogenous B and T lymphoid stem cells was determined by exposing host rats to between 4.5 and 10 Gy of gamma irradiation before repopulation with genetically marked marrow. The results depended on whether chimerism was assayed before day 50 or after day 100. At early times, a radioresistant T stem cell was indicated, whose activity waned later. Thus committed T stem cells of rats carry moderate amounts of W3/13 antigen and are more radioresistant but less permanently chimeragenic than the stem cells that regenerate B lymphocytes.     

Kinetics of human hemopoietic cells after in vivo administration of granulocyte-macrophage colony-stimulating factor.

The kinetic changes induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) on hemopoietic cells were assessed in physiological conditions by administering GM-CSF (8 micrograms/kg per d) for 3 d to nine patients with solid tumors and normal bone marrow (BM), before chemotherapy. GM-CSF increased the number of circulating granulocytes and monocytes; platelets, erythrocytes, lymphocyte number, and subsets were unmodified. GM-CSF increased the percentage of BM S phase BFU-E (from 32 +/- 7 to 79 +/- 16%), day 14 colony-forming unit granulocyte-macrophage (CFU-GM) (from 43 +/- 20 to 82 +/- 11%) and day 7 CFU-GM (from 41 +/- 14 to 56 +/- 20%). The percentage of BM myeloblasts, promyelocytes, and myelocytes in S phase increased from 26 +/- 14 to 41 +/- 6%, and that of erythroblasts increased from 25 +/- 12 to 30 +/- 12%. This suggests that GM-CSF activates both erythroid and granulomonopoietic progenitors but that, among the morphologically recognizable BM precursors, only the granulomonopoietic lineage is a direct target of the molecule. GM-CSF increased the birth rate of cycling cells from 1.3 to 3.4 cells %/h and decreased the duration of the S phase from 14.3 to 9.1 h and the cell cycle time from 86 to 26 h. After treatment discontinuation, the number of circulating granulocytes and monocytes rapidly fell. The proportion of S phase BM cells dropped to values lower than pretreatment levels, suggesting a period of relative refractoriness to cell cycle-active antineoplastic agents.     

Minimal disease detection and confirmation in hematologic malignancies: combining cell sorting with clonality profiling

BACKGROUND: In this study we demonstrate the technical application of flow cytometry and cell sorting combined with gene-rearrangement clonality profiling to detect and confirm minimal disease in 2 leukemia and 2 lymphoma cases. METHODS: Specimens with low percentages (0.05%-5%) of abnormal lymphoid populations were identified by flow cytometry. The abnormal lymphoid populations were sorted by flow cytometry, and the purified tumor populations along with unsorted fractions were subsequently analyzed for the presence of clonal gene rearrangements by PCR and fluorescence-based capillary electrophoresis fragment analysis. RESULTS: In 3 cases, distinct clonality profiles could be detected in the purified tumor cell fraction, and suspicious amplicons of identical sizes were detected among the polyclonal backgrounds in the unsorted specimens. For 1 patient, a monoclonal signal was detected in the sorted tumor cell fraction but not in the unseparated bone marrow specimen containing 0.05% abnormal lymphoblasts. A subsequent bone marrow specimen containing 4.8% recurring leukemia cells tested positive with a clonality profile that matched the previous profile in the sorted cell population. CONCLUSIONS: The described method integrating 2 technologies allows genotypic confirmation of an aberrant population detected by immunophenotype to increase diagnostic certainty. This strategy provides a sensitive tool for disease monitoring without the need for patient-specific primer design and assay optimization required for quantitative PCR analysis.     

Expression of myeloid differentiation antigens on myeloblasts in bone marrow of patients with myelodysplastic syndromes

Bone marrow myeloblasts in 15 patients with myelodysplastic syndromes were quantitated with monoclonal antibodies using the immunoalkaline phosphatase technique. Positive blasts were identified in 7 of the 15 cases with at least one of three antibodies reactive with acute myelomonocytic leukaemic cells (PMN-6, PMN-29, AML 2-23) which were non-reactive with normal myeloblasts. In 5 of these cases increased PM-81 positivity was associated with expression of at least one of the other antigens (PM-81 antibody reacts with all types of acute myeloid leukaemic cells and a certain percentage of normal myeloblasts). The data suggest an aberration of myeloid differentiation in myelodysplastic syndromes which is reflected in altered surface marker features.     

类白血病反应动态观察1例及其分类分析

本文报道类风湿关节炎病人在药物治疗中发生类白血病反应1例。患者在抗风湿药物治疗中出现腹泻、发热及粒细胞缺乏等表现。外周血象表现为核左移，见2％早幼粒细胞，骨髓细胞学检查发现早幼粒细胞迭41％。分析病例及文献，本例属于急性早幼粒细胞型类白血病反应，也符合骨髓类白血病反应的特点。     

Multiplication of human fetal myelomonocytic precursors in medium conditioned by acute myelocytic leukemia cells treated by 12-O-tetradecanoyl phorbol 13-acetate (TPA)

An 8-21 fold multiplication of myeloid cells and macrophages was observed in tissue culture from human fetal bone marrow. Proliferation of the cells was triggered by medium conditioned by acute myelocytic leukemic cells exposed to 12-O-tetradecanoyl phorbol 13-acetate (TPA). The medium was designated as TPA-treated cell conditioned medium (TPACCM). The cycle of events in fetal bone marrow cell culture began with a sharp decrease in the total number of cells. At this juncture a predominance of primitive macrophage-like cells positive for macrophage markers was present in the culture. The process of multiplication began with rapid proliferation of promyelocytes. Simultaneous proliferation of granulocyte-macrophage (GM) colony forming cells (CFC) indicated that at least some of the promyelocytes might act like CFC. The absolute number of committed CFC then increased 5-11 fold, the number that approximated a total multiplication of FBM cells. The proliferation continued with the culture containing some blast cells and showing simultaneous differentiation of the cells into mature granulocytes and macrophages. The cells with macrophage markers persisted throughout the culture period. To determine more precise definition of the role of primitive macrophages and promyelocytes in FBM cell multiplication, experiments with purified fractions of these cells have to be done. TPACCM purification and isolation of active substances is also suggested. These investigations might result in obtaining a pool of BM cells in vitro suitable for BM transplantation.     

Normalization of bone marrow aspirates for hemodilution in flow cytometric analyses

The use of flow cytometric enumeration of blasts in bone marrow aspirates has been of limited value in situations where blood contamination of the specimen is present. Assessment of sequential pulls of bone marrow aspirates from the same patient show decreasing proportions of blasts that are detected in later specimens. To address this problem, the intensity of CD16 on maturing neutrophils was compared for bone marrow biopsies, peripheral blood, and bone marrow aspirates. A comparison between bone marrow biopsy and aspirate specimens from the same individuals showed similar proportions of neutrophils with mature phenotype in most, but not all pairs. Other cell populations (total mature lymphocytes, monocytes, neutrophils, and blasts) were also similar between the two specimen types, with one exception of a patient with myelodysplasia, exhibiting a unique blast population in the biopsy that was not evident in the aspirate. The proportion of mature myeloid cells expressing a mature neutrophil phenotype (high levels of CD16) was found to be 17% (±6.7, n=47) in trephine marrow biopsy specimens. In contrast, marrow aspirates contained more of the mature neutrophil phenotype (38%±16, n=33) with about 1/3 of the aspirates indistinguishable from biopsies. Using a simple formula to normalize the aspirate specimens to the average neutrophil composition of marrow biopsies, it was possible to correct for the dilutional effect of added blood to both normal bone marrow aspirates and aspirates with elevated blast counts. These results suggest three alternative means of circumventing the problem of blood dilution of marrow aspirate specimens. (1) Blast counts by flow cytometry can be obtained from disaggregated biopsy specimens. (2) A bone marrow aspirate can be assessed for the proportion of mature neutrophils present and only those with low proportions (<30%) of phenotypic mature neutrophils are considered adequate for blast counting. (3) The aspirates with high proportions of mature neutrophils may be normalized based on the proportion of dim CD16 maturing myeloid cells to a level observed in bone marrow biopsies (based on an average mature neutrophil composition). Such an approach for identifying the amount of hemodilution in each specimen may enhance the utility of flow cytometry in enumeration of blasts in bone marrow, especially in cases where myeloblast count is crucial for prognosis, such as myelodysplastic syndromes (MDS).     

Leukaemic promyelocytes and normal bone marrow cells release a passive sodium transport modifier (inhibitin)

Previous studies have shown that leukaemic immature cells, specifically promyelocytes but not mature leucocytes, contain and release an inhibitor of ouabain-insensitive sodium transport (inhibitin). In the present study, medium from cultured leukaemic promyelocytes significantly reduced the ouabain-insensitive sodium efflux from erythrocytes, whereas medium from the same cell line which had been made to differentiate did not have this effect. Culture medium from normal bone marrow cells (containing promyelocytes) also significantly (P less than 0.001) reduced ouabain-insensitive sodium efflux. These data suggest that inhibitin is secreted by primitive but not by mature leukaemic cells and normal bone marrow cells.     

42例苯中毒致严重再生障碍性贫血患者的临床特征

对４２例符合急性再生障碍性贫血（ＡＡＡ）诊断的苯中毒患者，采用ＳＳＬ方案治疗。按疗效将其分为ＡＡＡ－ａ组（６例）和ＡＡＡ－ｂ组（３６例），并与８例原发性ＡＡＡ和５０例原发性慢性再生障碍性贫血（ＣＡＡ）相比较。结果苯中毒ＡＡＡ－ａ组患者的骨髓增生度、有核细胞分裂指数、原始＋早幼粒细胞、原始＋早幼红细胞等反映骨髓造血功能的指标与原发性ＡＡＡ组相似（Ｐ＞０．０５），采用ＳＳＬ方案治疗无效，均在６个月内死亡；苯中毒ＡＡＡ－ｂ组上述反映骨髓造血功能指标明显高于原发性ＡＡＡ组及苯中毒ＡＡＡ－ａ组（Ｐ均＜０．０１），采用ＳＳＬ方案治疗均有效，缓解率达８８．９％。认为ＡＡＡ－ａ组应归入ＡＡＡ，而ＡＡＡ－ｂ组近似于ＣＡＡ中的ＳＡＡ－ＩＩ型。两组苯中毒致再生障碍性贫血患者骨髓造血功能和对ＳＳＬ方案治疗结果的不同，当属量的差异还是质的不同，值得进一步研究。作者根据骨髓造血的一些指标设计了分级记分分类法，ＳＡＡ－ＩＩ型积分均≥４分，ＡＡＡ积分均≤３分，认为该分类法可作为两者鉴别的一种手段。     

免疫磁珠选择性清除人异基因反应性T细胞的实验研究

本研究用磁性细胞分离系统清除CD25^＋和CD69^＋人同种异体反应性T细胞，为减轻异基因骨髓移植后的移植物抗宿主病探索新的手段、对健康供者外周血单个核细胞与白血病缓解病人的骨髓单个核细胞进行单向混合淋巴细胞培养。3天后，通过磁性细胞分离系统清除CD25^＋和CD69^＋的同种异体反应性T细胞；通过^3H-TdR掺入实验，观察处理后的供者外周血单个核细胞对同一病人的骨髓单个核细胞及无关者外周血单个核细胞的反应性．结果表明：与未清除组相比，清除后供者细胞对同一病人抗原的反应性降低50％，但保留了大部分对无关者抗原的反应性。结论：在体外反应中清除同种异体反应性T细胞可降低对刺激抗原反应，同时保留大部分对无关者抗原的反应。     

Evaluation of a new screening system for enteric pathogens

The two-hour Rapid SST strip (DMS Laboratories, Inc.) was compared with our standard screening system (triple sugar iron agar, lysine iron agar, and urea) for enteric pathogens. We tested 50 stock cultures of enteric pathogens and 213 stool cultures received in the Barnes Hospital Clinical Microbiology Laboratory over a two-month period. All enteric pathogens from the stock cultures and clinical specimens were identified correctly with the Rapid SST system. More false-positive reactions were observed with the Rapid SST system (34%) than with the conventional system (23%). However, the costs associated with using both systems were equivalent and the test results were available one day faster with the Rapid SST system. Thus, the Rapid SST is a rapid, accurate, and cost-effective method for screening stool specimens for enteric pathogens.     

骨髓增生异常综合征免疫表型研究进展

骨髓增生异常综合征（MDS）是一组血液系统异质性的疾病,表现为骨髓原始细胞的形态和数目异常,无效造血及不同程度的外周血细胞减少并易转变为急性髓系白血病。本文就MDS疾病的骨髓形态学检查,骨髓活检,染色体核型分析,免疫分型等问题,特别是流式细胞术免疫分型技术检测对MDS诊断和预后意义的研究进展作一综述。     

Ferumoxides–protamine sulfate is more effective than ferucarbotran for cell labeling: implications for clinically applicable cell tracking using MRI

The use of superparamagnetic iron oxide (SPIO) for labeling cells holds great promise for clinically applicable cell tracking using magnetic resonance imaging. For clinical application, an effectively and specifically labeled cell preparation is highly desired (i.e. a large amount of intracellular iron and a negligible amount of extracellular iron). In this study we performed a direct comparison of two SPIO labeling strategies that have both been reported as efficient and clinically translatable approaches. These approaches are cell labeling using ferumoxides–protamine complexes or ferucarabotran particles. Cell labeling was performed on primary human bone marrow stromal cells (hBMSCs) and chondrocytes. For both cell types ferumoxides–protamine resulted in a higher percentage of labeled cells, a higher total iron load, a larger amount of intracellular iron and a lower amount of extracellular iron aggregates, compared with ferucarbotran. Consequently, hBMSC and chondrocyte labeling with ferumoxides–protamine is more effective and results in more specific cell labeling than ferucarbotran. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitative determination of non-haem iron and ferritin iron in bone marrow using flameless atomic absorption spectrophotometry. A comparative study on the cytological and chemical determination of the bone marrow iron content

A method for non-haem iron analysis in bone marrow aspirates using graphite furnace atomic absorption spectrophotometry has been developed. Bone marrow aspirates were obtained from patients with various disorders. A good correlation is observed between chemical and cytological assessment of total non-haem iron in bone marrow. An intra-assay coefficient of variation of 9.0% was observed. The ferritin-iron concentration was also determined and a CVduplo of 11% was found. The ferritin iron concentration increased with an increasing total iron content until saturation of ferritin appeared to be reached at about 3 g ferritin per kg protein. It was concluded that the quantitative determination of bone marrow iron can be of value in the diagnosis and investigation of both hypo- and hyper-ferraemic disorders.     

In vivo evidence for the functional heterogeneity of transferrin-bound iron. III. Studies of transferrin at high and low iron saturation.

The functional heterogeneity of the transferrin iron pool of rats was studied by means of selective radioiron labeling of transferrin at high and low iron saturations. A sample of iron-poor plasma transferrin brought to 90 per cent iron saturation by the addition of 59Fe-nitrilotriacetate was mixed with a similarly labeled plasma sample of 55Fe-transferrin at 10 per cent iron saturation. The mixture was injected intravenously into groups of normal rats which were killed after 30 minutes, 3, and 24 hours for measurement of the distribution of the 59Fe and 55Fe in various tissues. 59Fe from diferric transferrin disappeared more rapidly from plasma and was preferentially removed by red blood cells, bone marrow, liver, and spleen. This phenomenon was most apparent at 30 minutes and 3 hours with little difference in the distribution of 59Fe and 55Fe at 24 hours. These studies add further support for the Fletcher-Huehns hypothesis of the functional heterogeneity of the transferrin iron pool in the rat.