Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: II. An efficient method of monitoring chromosomal damage in the rat.

We have evaluated a flow cytometric method that allows assessment of micronucleated reticulocytes (MN-RETs) in microliter quantities of peripheral blood and compared results using this assay with those of established microscopic methods of scoring bone marrow and peripheral blood from rats treated with well-characterized genotoxic agents. Young reticulocytes (RETs) are labeled with FITC-anti-CD71 (transferrin receptor) and micronuclei with propidium iodide (with RNase treatment). Red blood cells parasitized with Plasmodia serve as a calibration standard for DNA content. Microscopic scoring used acridine orange (AO) staining of methanol-fixed slides or supravital AO staining. The effect of the rat spleen on the parameters evaluated was determined by comparing age- and sex-matched normal and splenectomized rats treated with cyclophosphamide, cis-platin, or vinblastine under treatment conditions that established a steady-state frequency of MN-RETs in the bone marrow and peripheral blood compartments. The data demonstrate the sensitivity and reproducibility of the flow cytometric assay in the Sprague-Dawley rat, and comparative studies using identical blinded samples at multiple laboratories show that inter- and intra-laboratory reproducibility is much higher with the flow method than with the microscopic methods currently employed for regulatory studies. A significant effect of splenic selection against genotoxicant-induced MN-RETs was observed with each of the three scoring methodologies, despite the fact that the flow and supravital AO techniques restrict analysis to the youngest fraction of RETs. The high precision of flow-based measurements also demonstrated a slight but statistically significant level of selection against spontaneously arising MN-RET. Despite these spleen effects, assay sensitivity for blood-based analyses was maintained by the flow method as it was shown to have superior counting statistics, lower variability, and higher sensitivity than manual scoring. The data suggest that flow cytometric assessment of micronucleus induction can be integrated into routine toxicity testing, eliminating the need for a separate bioassay.     

Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes IV: An Index of Chromosomal Damage in the Rhesus Monkey (Macaca mulatta)

We report evaluation in rhesus monkeys of a flow cytometricprocedure (MicroFlow) that has previously been shown to allowassessment of micronucleated reticulocytes (MN-RETs) in theperipheral blood of rats and dogs. Reticulocytes (RETs) werelabeled with anti-CD71-fluorescein isothiocyanate, DNA was stainedwith propidium iodide using RNase treatment, and anti-CD61-phycoerythrinwas used to reduce interference from platelets. Flow cytometricdata were compared with microscopic scores of peripheral bloodand bone marrow using standard acridine orange staining. A singleiv administration of cyclophosphamide (CP, 5 mg/kg) inducedan approximately 10-fold increase in blood MN-RET frequency,with the peak occurring 2 days after administration. After dailyCP treatment to approximate a steady-state condition, the frequencyof MN-RETs in peripheral blood was approximately 25% of thatin bone marrow, indicating strong selection against MN-RETs.Nonetheless, CP-treated animals exhibited markedly elevatedblood MN-RET values (2.45–3.99%, n = 3; compared to amean baseline of 0.12%, n = 6). These measurements closely reflectedthe increased frequencies observed in the bone marrow compartment(Spearman correlation coefficient = 0.9856, n = 6). These datasuggest that MN-RET measurements in blood are suitable for assessingchemical-induced chromosomal damage and can be readily integratedinto routine toxicity tests, allowing genotoxicity data to beobtained as an integral part of toxicity evaluations. Microscopy-basedscoring is challenging due to the low frequency of RETs andMN-RET in monkeys, but sufficient numbers of cells are easilyscored with the flow cytometric procedure.     

Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: I. Intra- and interlaboratory comparison with microscopic scoring.

Accumulating evidence suggests that reticulocytes (RETs) in the peripheral blood of rats may represent a suitable cell population for use in the micronucleus assay, despite the ability of the rat spleen to selectively remove micronucleated erythrocytes from the peripheral circulation. To evaluate the analytical performance of a previously described flow cytometric method (Torous et al., 2003, Toxicol. Sci. 74, 309-314) that may allow this assay to be conducted using peripheral blood in lieu of bone marrow sampling, we compared the sensitivity and performance characteristics of the flow cytometric technique with two established microscopy-based scoring methods. Peripheral blood samples from single Sprague-Dawley rats treated for 6 days with either vehicle or cyclophosphamide were prepared in replicate for scoring by the three methods at different laboratories. These blood-based measurements were compared to those derived from bone marrow specimens from the same animals, stained with acridine orange, and scored by microscopy. Through the analysis of replicate specimens, inter- and intralaboratory variability were evaluated for each method. Scoring reproducibility over time was also evaluated. These data support the premise that rat RETs harvested from peripheral blood are a suitable cell population to assess genotoxicant-induced micronucleus formation. The interlaboratory comparison provides evidence of the general robustness of the micronucleus endpoint using different analytical approaches. Furthermore, data presented herein demonstrate a clear advantage of flow cytometry-based scoring over microscopy-significantly lower inter- and intralaboratory variation and higher statistical sensitivity.     

Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy.

A flow cytometric technique for scoring the incidence of micronucleated reticulocytes in rat peripheral blood was compared to a standard microscopy-based procedure. For these studies, groups of five male Sprague-Dawley rats were treated with vehicle or a broad range of chemical genotoxicants: 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, vincristine, methylaziridine, acetaldehyde, methyl methanesulfonate, benzene, monocrotaline, and azathioprine. Animals were treated once a day for up to 2 days, and peripheral blood was collected between 24 and 48 h after the final administration. These samples were processed for flow cytometric scoring and microscopy-based analysis using supravital acridine orange staining, and the percentage of reticulocytes and micronucleated reticulocytes was determined for each sample. The resulting data demonstrate good agreement between these scoring methodologies, although careful execution of the flow cytometric method was found to enhance the micronucleus assay by reducing both scoring time and scoring error. These data add further support to the premise that the peripheral blood compartment of rats can be used effectively to detect genotoxicant-induced micronuclei.     

The quantification of reticulocyte maturation and neocytolysis in normal and erythropoietin stimulated rats

A technique has recently been proposed for obtaining the reticulocyte (RET) age distribution from the flow cytometric reticulocyte count. It allows for a quantitative characterization of reticulocyte dynamics. In this work this technique was applied to characterize the blood, bone marrow and spleen reticulocytes in homeostatic and erythropoietically stimulated rats in order to determine the reticulocyte maturation times in the bone marrow and blood; and to confirm the presence of ineffective erythropoiesis (neocytolysis). The latter was done by comparing the reticulocyte removal rate from blood with bilirubin formation after erythropoiesis stimulation. A single subcutaneous dose (4050 IU/kg) of recombinant human erythropoietin (rHuEPO) was administered to rats, then their reticulocytes were stained with thiazole orange and the distribution of the fluorescent signal measured using flow cytometry. The obtained signal distribution of the reticulocytes was transformed to the age distribution and a set of basic parameters reflecting reticulocyte dynamics was determined. Bilirubin concentrations were measured to directly assess the presence of reticulocyte irreversible removal. The bilirubin formation was found to be considerably modulated by rHuEPO and corresponded well to the determined reticulocyte removal rate. The initial increase and subsequent decrease of the reticulocyte maturation time in blood was quantitated and directly linked with RET mobilization from the bone marrow. A substantial number (60%) of reticulocytes is sequestrated during homeostasis in rats. This number increases and then decreases after rHuEPO administration, as also reflected by bilirubin formation. Flow cytometry seems to be an excellent method for studying RET dynamics and the presence of young RBC neocytolysis. Copyright © 2014 John Wiley & Sons, Ltd.     

Zidovudine plus sulfamethoxazole-trimethoprim adversely affects B lymphocyte maturation in bone marrow of normal mice

Sulfamethoxazole-trimethoprim and zidovudine (AZT), drugs used often in combination in patients infected with HIV, were investigated for their effects on B cell development in a mouse model. BALB/c mice were randomized to receive oral doses of AZT, sulfamethoxazole-trimethoprim, or the combination via oral gavage for up to 28 days. Immune cell populations in the spleen, lung, and peripheral blood were examined, and toxicity to B lineage subtypes in the bone marrow was investigated by phenotypic analysis via flow cytometry. Pre-pro-B, pro-B, early pre-B, and late pre-B cells were assayed for apoptosis and analyzed for cell cycle profile. Total as well as B cell splenic and bone marrow cellularities were significantly decreased by using the drugs concomitantly, while B cell populations in the lungs and percentage in the peripheral blood were not affected. Combination therapy caused significant increases in apoptosis in B cells and granulocytes in the bone marrow, with the late pre-B cell population being the most depleted. The proliferative expansion and differentiation of early pre-B cells (B220+/CD43+/BP-1+/HSA+) to the late pre-B cell (B220+/CD43-/IgM-) stage was blocked, with early pre-B cells accumulating in the proliferative phases of the cell cycle. This apoptosis increase is likely due to elevated blood sulfamethoxazole concentrations that were observed in mice also receiving AZT. Concurrent sub-chronic administration of AZT and sulfamethoxazole-trimethoprim adversely affected B lymphocyte development in mouse bone marrow.     

Cytogenetic activity of methyl isocyanate in vivo in the mouse micronucleus test

The ability of methyl isocyanate (MIC) to induce mutagenic and cytotoxic effects in vivo in the mouse micronucleus test was evaluated by assessing the induction of micronuclei and depression of polychromatic erythrocytes in bone marrow and peripheral blood smears. Animals were exposed to MIC through intraperitoneal injection for 2 and 5 days in separate experiments, and bone marrow and peripheral blood were sampled 6 and 48 h after the last injection, respectively. MIC did not significantly increase the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) and micronucleated normochromatic erythrocytes (MN-NCE) in bone marrow and peripheral blood samples respectively in either twice or multiply treated mice. However, a dose-dependent depression in percentage PCE observed was significant. This indicates that MIC exposure led to the cytotoxic effect by inhibition of bone marrow cell proliferation.     

Saw palmetto extract induces nuclear heterogeneity in mice

Saw palmetto (SW), a phytotherapeutic compound used in the treatment of prostate disease, was examined for potential nuclear effects. SW extract was incorporated into a complete casein-based semisynthetic rodent chow at 0%, 0.1% and 1% SW. SW was fed to mice for 6 weeks, after which the mice received a single i/p injection of either the known genotoxic agent methyl methanesulfonate (MMS) in saline or just saline. Forty-eight hours after injection, blood and bone marrow were collected for flow cytometric analysis. A significant effect of MMS was observed in both male and female mice with respect to: an increase in nuclear heterogeneity in bone marrow cells as measured by the coefficient of variation of the G1 peak in a flow histogram (6.32 versus 4.8 in male mice, 7.0 versus 4.9 in female mice) and an increase in the number of micronucleated blood cells (3.4% versus 0.56% male mice, 3.1% versus 0.6 in female mice) indicating a positive genotoxic response. SW also appears to increase the heterogeneity of bone marrow nuclei in a dose dependent manner (0-5.1%, 0.1-5.5% and 1-5.7% in male mice, 0-5.7%, 0.1-6.0% and 1-6.2% in female mice) without a concomitant increase in blood cell micronuclei. These results indicate that SW is not genotoxic with respect to physical DNA damage and that the changes observed in the bone marrow are due to chromatin conformation modifications in the nuclei of in vivo treated mouse cells.     

Protective effects of total saponins from stem and leaf of Panax ginseng against cyclophosphamide-induced genotoxicity and apoptosis in mouse bone marrow cells and peripheral lymphocyte cells.

BACKGROUND: Cyclophosphamide (CP), commonly used anti-cancer, induces oxidative stress and is cytotoxic to normal cells. It is very important to choice the protective agent combined CP to reduce the side effects in cancer treatment. Ginsenosides are biological active constituents of Panax ginseng C.A. Meyer that acts as the tonic agent for the cancer patients to reduce the side effects in the clinic application. Because CP is a pro-oxidant agent and induces oxidative stress by the generation of free radicals to decrease the activities of anti-oxidant enzymes, the protective effects of the total saponins from stem and leaf of P. ginseng C.A. Meyer (TSPG) act as an anti-oxidant agent against the decreased anti-oxidant enzymes, the genotoxicity and apoptosis induced by CP was carried out. METHODS: The alkaline single cell gel electrophoresis was employed to detect DNA damage; flow cytometry assay and AO/EB staining assay were employed to measure cell apoptosis; the enzymatic anti-oxidants (T-SOD, CAT and GPx) and non-enzymatic anti-oxidant (GSH) were measured by the various colorimetric methods. RESULTS: CP induced the significant DNA damage in mouse peripheral lymphocytes in time- and dose-dependent manners, inhibited the activities of T-SOD, GPx and CAT, and decreased the contents of GSH in mouse blood, triggered bone marrow cell apoptosis at 6 and 12h. TSPG significantly reduced CP-induced DNA damages in bone marrow cells and peripheral lymphocyte cells, antagonized CP-induced reduction of T-SOD, GPx, CAT activities and the GSH contents, decreased the bone marrow cell apoptosis induced by CP. CONCLUSIONS: TSPG, significantly reduced the genotoxicity of CP in bone marrow cells and peripheral lymphocyte cells, and decreased the apoptotic cell number induced by CP in bone marrow cells. The effects of TSPG on T-SOD, GPx, CAT activities and GSH contents might partially contribute to its protective effects on CP-induced cell toxicities.     

Evaluation of genotoxicity of oral exposure to tetravalent vanadium in vivo

The trace element vanadium interacts with living cells, in which it exerts a variety of biological effects depending on its chemical form and oxidation state. Tetravalent vanadium was shown to affect several genotoxicity end-points in vitro, but its genotoxic potential in vivo is not elucidated. In this study, the genotoxic effects induced in vivo by subacute oral exposure to vanadyl sulphate (VOSO4), a tetravalent vanadium salt, were investigated. To this aim male CD1 mice were administered with VOSO4 in drinking water over the dose range 2-1000 mg/l for 5 weeks. The incidence of micronucleated blood reticulocytes was measured along treatment period. At the end of treatment, micronuclei in both blood reticulocytes and bone marrow polychromatic erythrocytes were determined; in addition, DNA lesions detectable by comet assay were assessed in marrow and testicular cells. Tissue distribution of vanadium at sacrifice was determined by atomic absorption spectrometry. Comet assays and the analysis of micronuclei in polychromatic erythrocytes did not reveal treatment related effects. A slight increase in micronucleated reticulocytes, with no relationship with the administered dose, was observed in some treated groups. The determination of vanadium content in kidney, liver, spleen, bone, stomach, small intestine and testis highlighted low internal exposure, especially in soft tissues. Overall, data indicate scarce bioavailability for orally administered tetravalent vanadium, and lack of significant genotoxic potential in vivo.     

Pre-bled-young-rats in genotoxicity testing: a model for peripheral blood micronucleus assay

Previously we have reported that prior bleeding increases the sensitivity of micronucleus (MN) assay in rats (Vikram et al., 2007 a). Rat peripheral blood micronucleus (PBMN) assay is generally not considered as reliable method for the assessment of clastogenic potential of test chemicals due to selective elimination of micronucleated cells from the circulation. The present study is aimed to evaluate the sensitivity of pre-bled-young-rat model in detecting genotoxins having different mechanism of action. In the present study, young male Sprague-Dawley (SD) rats (21-24 days old, weighing 60+/-5 g) and swiss mice (24-28 days old, weighing 15+/-2g) were used. Streptozotocin (STZ, 50mg/kg), Methotrexate (MTX, 10mg/kg), N-nitrosodiethylamine (DEN, 200mg/kg), Quercetin (QC, 50mg/kg) and Zidovudine (AZT, 400mg/kg) were used in the present experiment. Effect of prior bleeding time (0, 2, 6, 12 and 24h) on the kinetics of MN formation with STZ and AZT was studied and 36 h post chemical exposure was found to be the most suitable time point for sample collection if prior bleeding time was 0, 2 and 6h. Further, the impact of prior bleeding (2h) on the kinetics of MN formation in the bone marrow was evaluated with STZ and maximum MN frequency was observed after 24h. The area under curve (AUC) analysis proves that prior bleeding leads to significant increase in the incidence of micronucleated reticulocytes (RETs) in the peripheral blood as compared to respective non-bled controls. Out of five tested chemicals AZT and STZ induced significant increase in the MN frequency in non-bled animals while at the same dose MTX, AZT, QC and STZ induced significant increase in MN frequency in the pre-bled-young-rats employing PBMN assay as the end point. Positive results with MTX, AZT, QC, STZ and negative results with DEN demonstrate both the sensitivity and specificity of pre-bled-young-rat model in the screening of chemicals for genotoxicity using PBMN assay as the end point.     

Reduction of cyclophosphamide-induced DNA damage and apoptosis effects of ginsenoside Rb(1) on mouse bone marrow cells and peripheral blood leukocytes

The present study investigated the protective effects of ginsenoside Rb₁ (GRb₁) against genotoxicity induced by cyclophosphamide (CP). Single cell gel electrophoresis, flow cytometry assay with annexin V-FITC/propidine iodide (PI) and acridine orange (AO)/ethidium bromide (EB) staining assay were employed to measure DNA damage and cell apoptosis, respectively. The activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GPx) and the malondialdehyde (MDA) content were also investigated by a number of colormetric methods. The results showed that the CP produced significant DNA damage and cell apoptosis in mouse bone marrow cells or peripheral blood leukocytes, markedly inhibited the activities of T-SOD and GPx, and markedly increased the MDA content. GRb₁ significantly inhibited DNA damages and cell apoptosis in mouse bone marrow cells or peripheral blood leukocytes induced by CP and antagonized the reduction of CP-induced T-SOD and GPx activities, and inhibited the increase in MDA content induced by CP. The anti-tumor study of GRb₁ showed that GRb₁ did not affect the anti-tumor activities of CP. In conclusion, GRb₁ had significant protective effects against DNA damage and apoptosis induced by CP.     

Protective effects of ginsenoside Rg<Subscript>3</Subscript> against cyclophosphamide-induced DNA damage and cell apoptosis in mice

Despite the significant anti-tumor activities, cyclophosphamide (CP) also shows cytotoxicity to normal cells. In order to explore the protective effects of drugs against CP-induced adverse effects, 20(S)-ginsenoside Rg₃ was tested for its possibly protective activities on CP-induced DNA damage and cell apoptosis in mouse bone marrow cells or peripheral lymphocyte cells. In the current study, the alkaline single cell gel electrophoresis (comet assay), flow cytometry assay with annexin V-FITC/PI and AO/EB staining assay were employed to measure DNA strand breakage and cell apoptosis, respectively. The activities of SOD and GPx and the contents of MDA were also tested by the various colormetric methods. The results showed that CP at a dose of 100 mg/kg, i.p. significantly caused DNA damages in both mouse bone marrow cells and peripheral lymphocyte cells, and markedly inhibited the activities of GPx and SOD and increased MDA contents in mouse blood. Moreover, CP at a dose of 200 mg/kg, i.p. triggered apoptosis in mouse bone marrow cells. On the other hand, 20(S)-ginsenoside Rg₃ orally administered at a dose of 20 mg/kg to the animals once a day for 2 days significantly inhibited CP-induced DNA damages in mouse bone marrow cells and peripheral lymphocyte cells, decrease the apoptotic numbers of bone marrow cells, antagonized the reduction of the activities of SOD and GPx, and the increase in MDA contents. In conclusion, 20(S)-ginsenoside Rg₃ showed the significant protective effects on CP-induced cell DNA damage and apoptosis. These effects might be partially attributed to its protective actions against CP-induced oxidative stress.     

免疫组织化学法和流式细胞术检测非小细胞肺癌骨髓微转移

目的 流式细胞和免疫组织化学技术检测非小细胞肺癌骨髓微转移状况的差异比较.方法 选取2004年3月至2007年5月我院手术治疗的非小细胞肺癌160例,术前未经化疗及放疗,术中抽取肋骨骨髓,分别应用流式细胞技术和免疫组化法检测骨髓中阳性细胞表达率.结果 160例非小细胞肺癌患者免疫组化检测骨髓转移阳性率为30.0％(48例),流式细胞术检测骨髓转移阳性率为34.4％(55例).112例免疫组化检查阴性的患者中,流式细胞术检测发现12例患者骨髓中存在微转移;而48例免疫组化检查阳性的患者中,有5例流式细胞术检测为阴性.结论 流式细胞术和免疫组化技术都可检测非小细胞肺癌骨髓微转移,流式细胞技术有着更高的敏感性和检测效率.     

In vivo genotoxicity assessment of acrylamide and glycidyl methacrylate

Acrylamide (ACR) and glycidyl methacrylate (GMA) are structurally related compounds used for making polymers with various properties. Both chemicals can be present in food either as a byproduct of processing or a constituent of packaging. We performed a comprehensive evaluation of ACR and GMA genotoxicity in Fisher 344 rats using repeated gavage administrations. Clastogenicity was measured by scoring micronucleated (MN) erythrocytes from peripheral blood, DNA damage in liver, bone marrow and kidneys was measured using the Comet assay, and gene mutation was measured using the red blood cell (RBC) and reticulocyte Pig-a assay. A limited histopathology evaluation was performed in order to determine levels of cytotoxicity. Doses of up to 20 mg/kg/day of ACR and up to 250 mg/kg/day of GMA were used. ACR treatment resulted in DNA damage in the liver, but not in the bone marrow. While ACR was not a clastogen, it was a weak (equivocal) mutagen in the cells of bone marrow. GMA caused DNA damage in the cells of bone marrow, liver and kidney, and induced MN reticulocytes and Pig-a mutant RBCs in a dose-dependent manner. Collectively, our data suggest that both compounds are in vivo genotoxins, but the genotoxicity of ACR is tissue specific.     

The increase of endothelial progenitor cells in the peripheral blood: a new parameter for detecting onset and severity of sepsis

Sepsis is a clinical syndrome characterized by non-specific inflammatory response with evidence of profound changes in the function and structure of endothelium. Recent evidence suggests that vascular maintenance, repair and angiogenesis are in part mediated by recruitment from bone marrow (BM) of endothelial progenitor cells (EPCs). In this study we were interested in whether EPCs are increasingly mobilized during sepsis and if this mobilization is associated with sepsis severity. Our flow cytometry data demonstrate that in the CD34+ cell gate the number of EPCs in the blood of patients with sepsis had a four-fold increase (45 +/- 4.5% p < 0.001) compared to healthy controls (12 +/- 3.6%) and that this increase was already evident at 6 hours from diagnosis (40.6 +/- 4.2 percent), reaching its maximum at 72 hours. Also the percentage of cEPCs identified in the patients with sepsis (35 +/- 4.6% of the CD34+ cell) was statistically different (p < 0.001) compared to that found in the blood of patients with severe sepsis (75 +/- 4.9%). In addition, we proved that at six hours after sepsis diagnosis, VEGF, CXCL8 and CXCL12 serum levels were significantly higher in septic patients compared to healthy volunteers 559 +/- 82.14 pg/ml vs 2.9 +/- 0.6 (p < 0.0001), 189.8 +/- 67.3 pg/ml 15 vs 11.9 +/- 1.6 (p = 0.014) and 780.5 +/- 106.5 pg/ml; vs 190.2 +/- 71.4 (p < 0.001). Our data suggest that the cEPC evaluation in peripheral blood, even at early times of diagnosis, in patients with sepsis can be envisaged as a valuable parameter to confirm diagnosis and suggest further prognosis.     

弥漫大B细胞淋巴瘤骨髓侵犯形态学及免疫表型特点分析

目的 探讨弥漫大B细胞淋巴瘤（DLBCL）骨髓侵犯形态学及免疫表型特点,为临床DLBCL侵犯骨髓的准确诊断提供一定的科学依据。方法 收集2014年6月—2019年6月我院收治的59例DLBCL患者的骨髓组织和外周血,分析DLBCL患者的骨髓细胞形态学、骨髓病理学、流式细胞学、荧光原位免疫杂交及其他相关检测的结果与临床特征。结果 59例发生骨髓侵犯的DLBCL患者多数表现为白细胞增高、贫血、血小板减少,有43例（72.88%）外周血中可见数量不等的瘤细胞。59例患者骨髓涂片中瘤细胞占有核细胞比例≥10％的有31例（52.54%）。瘤细胞侵犯骨髓方式以弥漫性增生为主,造血组织明显减少或缺乏。免疫组化染色及流式细胞学检查示,淋巴瘤细胞 CD20、CD19、CD79b及 cCD79a均呈阳性表达,部分患者表达 HLA-DR、CD22、sIgM、CD43、CD10、FMC7及 CD123,均不表达CD38、TdT、CD103、CD25、CD3、CD2、MPO、CD33、CD13、CD7及CD56。12例患者进行FISH检测均未见C-myc基因重排。结论 骨髓形态学、病理学并结合免疫表型检测是DLBCL患者准确诊断的重要依据。     

Primary bone marrow B-cell lymphoma: report of four cases

Bone marrow involvement is infrequent at presentation in cases of diffuse large B-cell lymphoma. We report four adult patients with diffuse large B-cell lymphoma in whom bone marrow involvement with hematologic manifestations was the predominant clinical feature at presentation. Three patients presented with a leukoerythroblastic blood picture and one with pancytopenia. In each case, the unusual hematologic manifestations, with bone marrow replacement and the presence of immature forms in the peripheral blood, led to consideration of alternative hematologic diagnoses, including acute granulocytic leukemia in three cases and a myelodysplastic syndrome in one. The correct diagnoses were established by immunohistochemistry on formalin-fixed, paraffin-embedded bone marrow for two cases and by flow cytometry on aspirated bone marrow or peripheral blood lymphocytes for the other two. Diffuse large B-cell lymphoma should be considered in the differential diagnosis of unusual hematologic presentations, particularly in the elderly.     

Effects of BW245C, a prostaglandin dp receptor agonist, on systemic and regional haemodynamics in the anaesthetized rat

1. Prostaglandin D (DP) receptor agonists have been shown to induce hypotension in rat models, possibly via peripheral vasodilation. However, it is not known which tissues and organs are most responsive. 2. In the present study, BW245C, a DP receptor-selective agonist, was administered to Inactin (Sigma, St Louis, MO, USA)-anaesthetized rats. Animals received three serial i.v. infusions (17 min each) of either BW245C (escalating doses of 0.3, 3 and 30 microg/kg; n=6) or vehicle (6% ethanol in normal saline; n=6). Mean arterial pressure (MAP) and heart rate were monitored continuously and regional blood flow was determined by the radionuclide-labelled microsphere method at baseline and at the end of each infusion. 3. It was found that BW245C dose-dependently reduced MAP; blood flow increased in forelimb skeletal muscle and skin, resulting in decreases in the regional vascular resistance (RVR) of skeletal muscle to -6+/-13, -53+/-11 and -68+/-6% of baseline following 0.3, 3 and 30 microg/kg BW245C, respectively (P<0.05 vs vehicle treatment for the two higher doses), and skin to -29+/-8, -55+/-8 (P<0.05) and -30+/-16% of baseline, respectively. Relative to vehicle, blood flow and RVR for brain, heart, lung, liver, stomach and kidney were not significantly affected by BW245C. 4. These results demonstrate that the hypotension resulting from DP receptor activation in the rat is mediated primarily through vasodilation of arterioles of skeletal muscle independent of changes in blood flow to vital organs.     

Effects of nisoldipine on recovery of coronary blood flow, sarcoplasmic reticulum function and other biochemical parameters in post-ischaemic porcine myocardium

The effects of nisoldipine (0.1 micrograms/kg/min; n = 9) or its solvent (n = 9) were studied in pigs, in which left anterior descending coronary artery (LADCA) blood flow in both groups was reduced to 20% of baseline for 60 min and reperfused for 2 hr. Infusions were started at 30 min of ischaemia and lasted throughout reperfusion. In both groups, flow reduction abolished regional contractile function and caused similar decreases in the level of creatine phosphate (CP; by 70%) and the energy charge (from 0.91 to 0.69), mean arterial blood pressure (by 25%), LVdP/dtmax (by 30%) and cardiac output (by 30%). During ischaemia LADCA blood flow slightly increased (from 14 +/- 8 to 24 +/- 6 mL/min/100 g; P less than 0.05) in the nisoldipine-treated animals, resulting in an increase in CP to 91 +/- 24% of baseline and preventing further decreases in energy charge, as observed in the solvent-treated animals. After 2 hr of reperfusion in neither group return of contractile function of the post-ischaemic myocardium was observed. Post-ischaemic blood flow in the nisoldipine-treated pigs increased from 24 +/- 6 mL/min/100 g to 76 +/- 14 mL/min/100 g and from 19 +/- 6 mL/min/100 g to 41 +/- 6 ml/min/100 g in the solvent-treated animals (P less than 0.05) after 2 hr of reperfusion. Myocardial work was significantly higher in the nisoldipine-treated animals (111 +/- 15 mmHg.L/min as compared to 69 +/- 14 mmHg.L/min in the solvent-treated pigs after 2 hr of ischaemia). The energy charge of the post-ischaemic myocardium was similar for both groups (0.84 +/- 0.02 for the nisoldipine-treated and 0.83 +/- 0.03 for the solvent-treated animals). The rate of sarcoplasmic reticular Ca2+ uptake of the non-ischaemic segment of the nisoldipine-treated animals was 61% higher (P less than 0.05) than that of the solvent-treated animals. In the post-ischaemic myocardium similar rates of Ca2+ uptake were found in both groups, but the activities were markedly lower as compared to the non-ischaemic myocardium. It is concluded that nisoldipine increases blood flow during reperfusion, which may have been caused by coronary vasodilatation. However, attenuation of the "no-reflow" phenomenon also contributed, since more rapid rephosphorylation of ADP leading to an increase in CP during ischaemia may have preserved jeopardized cells. Moreover, nisoldipine increases the sarcoplasmic reticular Ca2+ pump activity independent of ischaemia, which may have contributed in reducing the Ca2+ overload.