Flow cytometric analysis of feline reticulocytes.

Hemolytic anemia was induced in five Domestic Shorthair cats (four adult males and one spayed female obtained from a breeding colony at Colorado State University, CO), and blood samples were analyzed from five other cats (two castrated male Domestic Shorthairs, one castrated male Domestic Longhair, one castrated male Persian, and one spayed female Siamese presented to the Veterinary Teaching Hospital at Colorado State University for miscellaneous problems). Blood samples taken from these cats had percentages of aggregate reticulocytes that ranged from 0% to 14.5% as determined by manual counting and were used to identify the best technique for staining cat reticulocytes for flow cytometric analysis. The best technique was mixing a blood sample (1/2,000 dilution) with 0.2 micrograms thiazole orange in 1 ml of diluent and incubating the mixture in the dark at room temperature for 30 to 60 minutes. The percentage of reticulocytes determined by flow cytometry correlated well (r = 0.88) with manually determined aggregate reticulocyte percentages; no significant differences were observed between the two techniques (P > 0.05). For the conditions used, punctate reticulocytes were not detected by flow cytometry. Samples with very high platelet numbers and very low packed cell volumes may show falsely elevated percentages of reticulocytes as determined by flow cytometry. The reproducibility of the flow cytometric technique was good; the coefficient of variation ranged from 4.8% to 17.9% in two samples with two different times of incubation. Staining of cat aggregate reticulocytes with thiazole orange and use of flow cytometric quantification is a reproducible technique that has a good correlation with the manual reticulocyte counting method.     

Clinical application of reticulocyte counts in dogs and cats.

Reticulocytes are anucleate immature red blood cells that contain a network of RNA, organelles, and mitochondria, which stain with supravital dyes. Both aggregate and punctate reticulocytes are present in domestic cats, and aggregate reticulocytes are used to assess the degree of regeneration in anemic dogs and cats. Multiple factors influence the degree of regenerative response to anemia. These factors include time of reticulocyte measurement, concurrent diseases, species, and ongoing therapy. Although many automated systems for reticulocyte enumeration exist, manual counts remain the gold standard in veterinary medicine.     

Evaluation of flow cytometric counting procedure for canine reticulocytes by use of thiazole orange.

An automated reticulocyte counting method that used a flow cytometer and the nucleic acid staining dye, thiazole orange, was developed. Anticoagulated (EDTA) blood specimens were suitable for flow cytometric reticulocyte counting when stored at 4 C for 96 hours after collection. Thiazole orange-stained samples were stable for 5.5 hours after staining when stored capped at 20 C and protected from light. Flow cytometric and manual microscopic reticulocyte counts were compared for counts in the 0.27 to 5.32% range (as determined by flow cytometry) and 0.10 to 4.90% range (as determined by 1 technician). Although the results of flow cytometric analysis generally correlated well (r = 0.821) with manual counts, there was poor correlation between the procedures for counts less than or equal to 2.0% (r less than or equal to 0.272). Linearity of flow cytometric counts over the range 0.27 to 14.46% was excellent (r = 0.999). Within-run precision of flow cytometric counts (% coefficient of variation [cv] = 3 to 5) was superior to manual microscopic counts obtained by one technician (% cv = 19 to 23) and to manual microscopic counts, which were an average of counts done by 3 technicians (% cv = 8 to 18). Comparable flow cytometric counts were obtained by counting 50,000 or 100,000 blood cells in the flow cytometer.     

Flow cytometric enumeration of reticulocyte in the peripheral blood from canine infected with Babesia gibsoni

Peripheral blood samples from dogs infected with Babesia gibsoni were analyzed by a flow cytometer for the percentage of reticulocytes after staining with a membrane-permeable fluorochrome, thiazole orange. Though thiazole orange has been reported to stain human reticulocytes and Plasmodium-infected erythrocytes, number of positive cells determined by the flow cytometry did not include that of erythrocytes infected with Babesia gibsoni. Analysis of 51 samples revealed a correlation coefficient of 0.96 as compared to the conventional determination by light microscopy. Separation of reticulocytes from Babesia gibsoni-infected erythrocytes by flow cytometry with or without the stain remained unresolved.     

Validation of the Sysmex XT‐2000iV hematology system for dogs,cats, and horses. I. Erythrocytes,platelets, and total leukocyte counts

Background: The Sysmex XT‐2000iV is a laser‐based, flow cytometric hematology system that has been introduced for use in large and referral veterinary laboratories. Objective: The purpose of this study was to validate the Sysmex XT‐2000iV for counting erythrocytes, reticulocytes, platelets, and total leukocytes in blood from ill dogs, cats, and horses. Methods: Blood samples from diseased animals (133 dogs, 65 cats, and 73 horses) were analyzed with the Sysmex XT‐2000iV and the CELL‐DYN 3500. Manual reticulocyte counts were done on an additional 98 canine and 14 feline samples and manual platelet counts were done on an additional 73 feline and 55 canine samples, and compared with automated Sysmex results. Results: Hemoglobin concentration, RBC counts, and total WBC counts on the Sysmex were highly correlated with those from the CELL‐DYN (r≥0.98). Systematic differences occurred for MCV and HCT. MCHC was poorly correlated in all species (r=0.33–0.67). The Sysmex impedance platelet count in dogs was highly correlated with both the impedance count from the CELL‐DYN (r=0.99) and the optical platelet count from the Sysmex (r=0.98). The Sysmex optical platelet count included large platelets, such that in samples from cats, the results agreed better with manual platelet counts than with impedance platelet counts on the Sysmex. Canine reticulocyte counts on the Sysmex correlated well (r=0.90) with manual reticulocyte counts. Feline reticulocyte counts on the Sysmex correlated well with aggregate (r=0.86) but not punctate (r=0.50) reticulocyte counts. Conclusion: The Sysmex XT‐2000iV performed as well as the CELL‐DYN on blood samples from dogs, cats, and horses with a variety of hematologic abnormalities. In addition, the Sysmex detected large platelets and provided accurate reticulocyte counts.     

赣南脐橙园蜘蛛优势种的季节动态及与气象因子的相关分析

赣南脐橙园蜘蛛已知13科38种。脐橙树冠层蜘蛛优势种有3种,其中草间钻头蛛(Hylyphantes graminicola)全年以5 - 6月及9 - 10月份的发生数量最大;斑管巢蛛(Clubiona deletrix)在7 - 10月份发生量较大,2 - 3月份的数量明显减少;鳞纹肖蛸(Tetragnatha squamata)在4月和12月份的种群数量较多,而6 - 9月份较少见到。地面层蜘蛛优势种有2种,其中草间钻头蛛的季节消长同样具有两个高峰期,但跨度较少,仅在6月份和10月份出现;拟水狼蛛(Pirata subpiraticus)种群数量在3月份达到最高峰,在12月份处于低谷。不同季节脐橙树冠层草间钻头蛛和斑管巢蛛的发生数量都与平均气温和日照时数呈显著或极显著正相关,而鳞纹肖蛸的季节发生数量与平均气温间为显著负相关;地面层拟水狼蛛的数量季节变动与相对湿度和降雨量间表现出极显著的正相关性。     

Hematology of healthy Florida manatees (Trichechus manatus)

Background: Hematologic analysis is an important tool in evaluating the general health status of free‐ranging manatees and in the diagnosis and monitoring of rehabilitating animals. Objectives: The purpose of this study was to evaluate diagnostically important hematologic analytes in healthy manatees (Trichechus manatus) and to assess variations with respect to location (free ranging vs captive), age class (small calves, large calves, subadults, and adults), and gender. Methods: Blood was collected from 55 free‐ranging and 63 captive healthy manatees. Most analytes were measured using a CELL‐DYN 3500R; automated reticulocytes were measured with an ADVIA 120. Standard manual methods were used for differential leukocyte counts, reticulocyte and Heinz body counts, and plasma protein and fibrinogen concentrations. Results: Rouleaux, slight polychromasia, stomatocytosis, and low numbers of schistocytes and nucleated RBCs (NRBCs) were seen often in stained blood films. Manual reticulocyte counts were higher than automated reticulocyte counts. Heinz bodies were present in erythrocytes of most manatees. Compared with free‐ranging manatees, captive animals had slightly lower MCV, MCH, and eosinophil counts and slightly higher heterophil and NRBC counts, and fibrinogen concentration. Total leukocyte, heterophil, and monocyte counts tended to be lower in adults than in younger animals. Small calves tended to have higher reticulocyte counts and NRBC counts than older animals. Conclusions: Hematologic findings were generally similar between captive and free‐ranging manatees. Higher manual reticulocyte counts suggest the ADVIA detects only reticulocytes containing large amounts of RNA. Higher reticulocyte and NRBC counts in young calves probably reflect an increased rate of erythropoiesis compared with older animals.     

Development of a technique for quantification of reticulocytes and assessment of erythrocyte regenerative capacity in birds

OBJECTIVE: To develop a reticulocyte classification scheme, optimize an avian reticulocyte staining protocol, and compare the percentages of reticulocyte types with polychromatophil percentage in blood samples from birds. SAMPLE POPULATION: Blood samples from a red-tailed hawk and 31 ill birds. PROCEDURES: A single blood sample obtained from a red-tailed hawk (Buteo jamaicensis) was used to optimize the staining protocol. For optimization of the staining protocol, 4 dilutions of whole blood with new methylene blue stain and 4 incubation times were evaluated. From samples submitted for avian CBCs, EDTA-anticoagulated whole blood samples from 31 ill birds were randomly selected and examined to compare polychromatophil and reticulocyte percentages. Reticulocyte staining was performed in all samples by use of a 1:3 (whole blood to new methylene blue) dilution with incubation for 10 minutes at room temperature (approx 22 degrees C); reticulocytes were assessed as a percentage of 1,000 RBCs by 2 independent observers. In Wright-Giemsa-stained blood smears, a polychromatophil percentage was similarly determined. RESULTS: 4 avian reticulocyte types were defined: ring-form reticulocytes, aggregate reticulocytes, and 2 subcategories of punctate reticulocytes. A reticulocyte-staining protocol was optimized. Interobserver and intraobserver variations in assessment of reticulocyte and polychromatophil percentages were not significant. A strong positive correlation (Spearman coefficient of rank correlation [rho] = 0.978) was identified between the percentage of polychromatophils and the percentage of ring-form reticulocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that quantification of ring-form reticulocytes provides an accurate assessment of erythrocyte regenerative capacity in birds.     

Flow cytometric evaluation of canine bone marrow differential cell counts

Abstract: Three flow cytometric techniques were evaluated for determination of differential cell counts on canine clinical bone marrow specimens. Techniques included staining bone marrow specimens with 2'7'-dichlo-rofluorescein (DCF) or 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and evaluation of forward-angle light scatter vs. side-angle light scatter plots. Flow cytometric evaluation of bone marrow cells stained with DCF failed to separate bone marrow cells into distinct cell populations. Staining with DiOC6 resulted in separation of bone marrow cells into populations of mature and immature erythroid cells, mature and immature myeloid cells, and lymphocytes. The scatter plot method resulted in identification of mature and immature erythroid cells, immature myeloid cells, metamyelocytes, and bands and segmenters. Lymphocytes could not be differentiated from mature erythroid cells by the scatter plot method. When the results of the DiOC6 method and the scatter plot method were compared with manual bone marrow differential cell counts, the scatter plot method had more similar mean values and higher correlation coefficients. The scatter plot method has the potential of providing rapid semiquantitative assessment of bone marrow differential cell counts in dogs for specimens that contain low numbers of lymphocytes.     

Evaluation of a novel haematology analyser for use with feline blood

A novel haematology analyser was evaluated for its use with feline samples. Complete blood cell counts, a five-part differential count, and reticulocyte numbers were determined, and the results compared with reference data. Coefficients of correlation, Passing–Bablok regression analysis and Bland–Altmann difference plots with biases and 95% limits of agreement are reported. Precision and linearity were also studied. The instrument demonstrated very low imprecision, and the tested range of linearity exceeded the reference ranges provided by the manufacturer. For all parameters except monocytes (r = 0.65), the analyser results correlated well with reference methods. Compared with the manual count of aggregated reticulocytes, the instrument showed good agreement with a positive bias. The optical platelet count correlated well with the manual chamber count. In conclusion the analyser was found to be highly reliable for the analysis of feline blood samples in a large veterinary laboratory.     

Tracer Dyes Food Green No. 4 and Food Blue No. 3 in Antibiotic and Chemotherapeutic Preparations for Intramammary Application Iv

Comparison has been made between Food Green No. 4 and Food Blue No. 3 as tracer dyes for indirect detection of the drugs in milk after intramammary application of a number of commonly used preparations containing antibiotics and chemotherapeutic agents.The tracer dyes had no effect on the stability of the preparations containing penicillin, penicillin ester, and sulphonamide (I, II, III, V and VI) ; after six months’ storage at room temperature, 75 to 100 per cent of the drugs could be recovered. The Oxytetracycline content in preparation IV recovered by 70 to 100 per cent after two months. There were great differences in the irritating effect of the various preparations, as determined by the cell counts of the milk, but the addition of tracer dye did not alter this effect. A direct relationship was demonstrated between concentrations of, on the one hand, tracer dye and, on the other hand, penicillin (preparations I, II, and III), Oxytetracycline (preparation IV) and sulphonamide (preparations V and VI) in milk from ten treated cows.In all quarter samples tracer dye could be demonstrated for just as long as the drugs, and often one or two milkings longer. Thus the experiments confirm the great certainty with which triphenylmethane dyes can reveal indirectly the presence of very small residues of antibiotics and sulphonamides in the milk after intramammary infusion.     

几种药剂对脐橙储藏期病害的防治效果比较研究

用7组药剂分别对纽荷尔脐橙做浸果处理,处理至135天,结果显示：百可得 咪鲜胺处理组烂果率7.78%,病情指数4.81,防效75.5%;咪鲜胺处理组烂果率13.33%,病情指数8.52,防效56.6%;百可得 抑霉唑处理组烂果率14.44%,病情指数8.15,防效58.48%;清水处理组烂果率30%,病情指数19.63。百可得 咪鲜胺处理组储藏效果最好,其次是咪鲜胺处理组和百可得 抑霉唑处理组。在脐橙的储藏保鲜中推荐使用百可得与咪鲜胺或抑霉唑混配或咪鲜胺单剂。     

黄原酸基高岭土对甲基橙的吸附性能研究

采用湿法工艺对高岭土进行改性,制备了黄原酸基高岭土（XK）。通过FT-IF、SEM分析对其进行表征,结果表明XK已成功合成;XK对甲基橙具有良好的吸附效果,其优点是处理后固液分离简便、快速。通过单因素试验得出最佳反应条件：温度25℃,p H值5-6、甲基橙初始质量浓度为200 mg/L、XK的用量为3.0 g/L、吸附30 min,甲基橙的去除率可达到99.86%,处理后残余甲基橙的浓度为0.282 4 mg/L,可达标排放。通过等温吸附模型研究,XK对甲基橙的吸附符合Freundlich等温吸附,说明XK对甲基橙的吸附是较为典型的单分子层吸附,且吸附容易进行。     

瓯柑橘皮和柑橘皮的抗缺氧、抗疲劳作用

在小鼠饲料中添加10%的柑橘皮和瓯柑橘皮,饲喂2周后进行密闭抗缺氧和负重游泳抗疲劳试验。抗缺氧试验结果表明,对照组、柑橘皮组和瓯柑橘皮组小鼠的缺氧窒息死亡时间分别为:19.53±3.21 m in,23.88±7.10,26.86±3.65;抗疲劳试验结果,对照组、柑橘皮组和瓯柑橘皮组小鼠游泳死亡时间分别为:3.99±0.75,3.86±1.82,5.32±1.20。结果证明橘皮和瓯柑柑橘皮对小鼠均有极显著抗缺氧作用(P0.01),而且瓯柑橘皮的作用显著大于柑橘皮(P0.05);瓯柑橘皮还有极显著的抗疲劳作用(P0.01),而柑橘皮无此作用(P0.05)。     

Quantitation of reticulated platelets in healthy dogs and in nonthrombocytopenic dogs with clinical disease

Background — Thrombocytopenia is a common disorder in dogs and development of an objective diagnostic assay to measure platelets newly released from bone marrow into the blood would provide a noninvasive way to predict megakaryocytopoiesis. Reticulated platelets are newly released platelets with increased concentrations of RNA that can be detected by flow cytometric analysis of blood stained with thiazole orange (TO).
Objectives — The goals of this study were to establish a reproducible method to quantitate reticulated platelets in dogs, to establish a reference interval for reticulated platelet percentages in healthy dogs, and to determine whether the percentage of reticulated platelets was nonspecifically increased in nonthrombocytopenic dogs with clinical disease.
Methods — Blood samples were obtained from healthy dogs and from nonthrombocytopenic dogs presented for a variety of disorders. An aliquot of whole blood was stained with TO and a phycoerythrin-labeled monoclonal antibody to platelet CD61, then analyzed by flow cytometry.
Results — The coefficients of variation were 7.8% to 15.6% (intra-assay precision) and 6.1% to 19.5% (interassay precision). Overnight storage for 18 to 26 hours, under variable conditions, resulted in an increase in the percentage of platelets staining with TO. The reference interval for reticulated platelets in the healthy control group was 0–4.3% (0–12,095/μL). No significant differences were found in the mean percentage of reticulated platelets or absolute concentration of reticulated platelets between control and affected dogs.
Conclusions — These studies demonstrate a reliable, noninvasive diagnostic assay for measurement of reticulated platelets in whole blood and provide a baseline for assessment of the clinical utility of the assay.     

Determination of differential cell counts in feline bone marrow by use of flow cytometry

OBJECTIVE: To evaluate the potential usefulness of 2 flow cytometric methods for determination of differential cell counts in feline bone marrow. SAMPLE POPULATION: 10 bone marrow specimens from client-owned cats. PROCEDURE: Bone marrow specimens were stained with 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and evaluated by use of flow cytometry. Differential counts were also determined by analysis of scatterplots of forward-angle versus side-angle light scatter of unstained specimens, obtained by use of flow cytometry (scatterplot method). Results of both flow cytometric methods were compared with differential cell counts determined by manually counting 1,000 cells on slides of Wright-stained smears. RESULTS: Staining with DiOC6 resulted in identification of mature and immature erythroid and myeloid cells and lymphocytes. Use of the scatterplot method resulted in identification of mature and immature erythroid and myeloid cells and metamyelocytes. However, to identify lymphocytes by use of the scatterplot method, bone marrow specimens were first labeled with an anti-major histocompatability class-II antibody. Comparison of results of the scatterplot method with manual counts yielded higher correlation coefficients and more similar mean values than did comparison of results of the DiOC6 method. Conclusions and Clinical Relevance: The scatterplot method provided more accurate and precise results than the DiOC6 method for determination of bone marrow differential cell counts in cats by use of flow cytometry. When combined with fluorescent labeling of lymphocytes, the scatterplot method has potential to provide rapid semiquantitative assessment of bone marrow differential cell counts in cats.     

Use of human recombinant erythropoietin for the treatment of nonregenerative anemia in a rough-toothed dolphin (Steno bredanensis).

Erythropoietin, a glycoprotein growth hormone that is produced primarily in the kidneys, promotes mitosis and survival of erythroid progenitors. The recent synthesis of the human form of the hormone by recombinant technology has provided a new therapeutic option, which is being used in both human and veterinary medicine for treatment of various anemias. A mature male rough-toothed dolphin, Steno bredanensis, was treated with human recombinant erythropoietin in an attempt to resolve a nonregenerative anemia. Two i.m. injections 48 hr apart were associated with an almost immediate increase in circulating immature reticulocytes, total reticulocytes, and nucleated erythrocytes. Over the next several weeks, the hematocrit, hemoglobin, and erythrocyte counts returned to normal, and the animal was subsequently released back into the wild. Endogenous erythropoietin concentrations were determined for this animal as well as three other conspecifics by an enzyme-linked immunosorbent assay for human erythropoietin. These measurements showed circulating erythropoietin concentrations (5-20+ mU/ml) similar to those of most other mammals. This study suggests that human recombinant erythropoietin can be safely and effectively used in this species and may have applicability to other cetacean species for the treatment of nonregenerative anemia. Caution should be exercised during long-term use because production of antibodies to human recombinant and endogenous erythropoietin may lead to potentially serious side effects.     

Use of flow cytometry for determination of differential leukocyte counts in bovine blood

A flow cytometric method was developed to perform differential leukocyte counts on bovine blood. Blood specimens from 50 healthy Holstein cows were analyzed by use of a flow cytometer. The method entailed diluting blood with phosphate-buffered, hypotonic saline solution containing acridine orange, and performing a step-wise, 3-parameter analysis on the bases of cell size, cellular granularity, and granulocyte fluorescence. Initially, proportions of monocytes, granulocytes, and lymphocytes were determined by creating appropriate windows on dot plots of cell size (determined by forward light scatter) vs cellular granularity (determined by the logarithm of side light scatter). Eosinophils were resolved by analysis of granulocytes as dot plots of logarithms of green vs red fluorescence ascribed to acridine orange. Proportions of eosinophils and neutrophils were computed from data so generated. Microclumps of platelets spuriously affected counts of some granulocytes, particularly eosinophils. Differential leukocyte counts determined by flow cytometry generally compared favorably with those obtained by use of the conventional microscopic method, using Wright-stained blood films. Mean neutrophil and eosinophil counts determined by the 2 methods did not differ significantly, but lymphocyte counts determined by flow cytometry were significantly higher than those determined by microscopy (P less than 0.01). Correlation coefficients for counts of neutrophils, eosinophils, and lymphocytes determined by the 2 methods ranged from 0.519 to 0.833. Correlation between monocyte counts was low (r = 0.147), although mean monocyte counts determined by the 2 methods did not differ significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo acridine orange human spermatozoa staining—A new perspective for RNA detection and spermatozoa morphology evaluation

Significant increases in male infertility and the still unresolved questions on the compatibility and interpretation of current methods in infertility diagnostics call for new protocols. Morphology, genome damage, RNA content and quantity are currently in practice as the major parameters in evaluation of sperm quality. However, results of various methods are not always in mutual concordance. In this study, in vivo acridine orange (AO) staining, which is presently in application in the estimation of genome damage in reticulocytes, was adjusted for spermatozoa staining. Ten men suffering from oligoasthenoteratozoospermia (OAT) and 10 healthy fertile men were analysed using in vivo AO staining. Microscopic analysis was performed by fluorescent and confocal fluorescent microscopy. Our results show that this method preserves spermatozoa membranes, which enables new insight into spermatozoa genome damage, RNA content in residual cytoplasm, damage of neck area with mitochondrion and tail pathology. The introduced method explains the difference between results of sperm DNA fragmentation assay and the globally used AO staining and opens new options for the development of automated systems. In conclusion, the results of our study offer (a) an innovative approach to the analysis of spermatozoa pathology, (b) enable localization and quantification of RNA in residual cytoplasm, (c) a significant contribution to research of aetiology of infertility in men, (d) open new perspectives for the automatization of sperm quality estimation and (e) improve the personalized approach in the selection of in vitro fertilization protocols.     

自动电位滴定法快速测定脐橙叶片中氯含量

建立了一种基于自动电位滴定技术的脐橙叶片中氯含量的快速检测方法。该法采用稀硝酸浸提脐橙叶片中氯,然后用自动电位滴定法快速测定溶液中氯含量,并考察了方法的测定条件、精密度和准确度。该方法的检出限为0.6249mg/L,加标回收率为94.85%～102.96%,相对标准偏差小于3.0%,故该法适于脐橙叶片中氯含量的快速分析。