Detection and quantitation of simulated anaerobic bacteremia by centrifugation and filtration.

Fresh human whole blood was inoculated with various anaerobic bacteria or with combinations of anaerobic, facultatively anaerobic, and aerobic microorganisms (3 to 28 microorganisms per ml). The seeded blood was then layered on a reduced Ficoll-Hypaque gradient (density, 1.093 g/ml) and centrifuged (400 X g) for 30 min at ambient temperature. The entire gradient (plasma, leukocytes, and Ficoll-Hypaque) was removed and filtered anaerobically through a 0.45-micron membrane filter. The filters were then placed on reduced chocolate Mueller-Hinton agar plates and incubated at 35 degrees C in humidified atmosphere containing 85% N2, 10% H, and 5% CO2 or in air containing 5% CO2. No statistically significant differences were detected between the numbers of microorganisms recovered (alone or in combination) by filtration and by direct culturing of the original inoculum. All organisms were detected within 30 h after filtration. This technique has excellent sensitivity.     

Detection of bacteria in blood by centrifugation and filtration.

Culture of blood is the most frequent means of diagnosing bacteremia. However, conventional blood culturing methods are slow in isolating bacteria. We developed a method for isolation of bacteria by centrifugation and filtration. Fresh human whole blood was inoculated with facultatively anaerobic and aerobic microorganisms (3 to 172 microorganisms per 5 ml). Seeded blood was then mixed with Ficoll-Hypaque (density, 1.149 +/- 0.002 g/ml) and centrifuged (386 x g) for 30 min at ambient temperature. The entire gradient (plasma, leukocytes, and Ficoll-Hypaque) was removed and filtered through a 0.22-micron membrane filter (Millipore). The filters were then placed on chocolate agar plates and incubated at 35 degrees C in a humidified atmosphere containing 5% CO2. For each bacterium tested, approximately 35 to 100% of the viable microorganisms were recovered when compared with control cultures (pour plates of seeded blood). All bacteria produced isolated colonies on filters after overnight incubation (18 h). This procedure may prove to be a more rapid method for isolating bacteria from clinical blood samples than the blood culture bottle technique.     

Evaluation and comparison of different blood culture techniques for bacteriological isolation of Salmonella typhi and Brucella abortus.

An experimental study was carried out to evaluate and compare various noncommercial methods of blood culture for the isolation of Salmonella typhi and Brucella abortus from fresh human blood samples that had been artificially inoculated with 1 to 50 microorganisms per ml of blood. The methods compared included the Ruiz-Castañeda blood culture, broth blood culture, leukocyte lysis and direct plating on agar (WBL-P), leukocyte lysis and filtration, Ficoll-Hypaque centrifugation and filtration, Ficoll-Hypaque centrifugation, and Ficoll-Hypaque centrifugation and leukocyte lysis methods. Results with the WBL-P technique showed that S. typhi was isolated in 18 h, and its recovery rate was 36.6% (calculated from the number of CFU recovered per milliliter versus the number inoculated). B. abortus was isolated in 48 h by the same technique, and its recovery rate was 48.8%. The isolation times for the other blood culture techniques were between 36 and 44 h for S. typhi and 66 h for B. abortus. The techniques which relied on filtering systems for the recovery of S. typhi and B. abortus performed poorly. The WBL-P technique for the isolation of S. typhi and B. abortus is faster than the other methods tested.     

Density gradient purification of human lymphocytes from contaminating trypomastigotes of Trypanosoma cruzi

Mixtures of normal human lymphocytes and T. cruzi trypomastigotes obtained from infected mice were centrifuged over Ficoll-Hypaque (FH) continuous and discontinuous gradients. Trypomastigotes were confined to the range 1.051–1.057 g/ml while lymphocytes ranged between 1.046 and 1.080 g/ml. Over 80% of the lymphocytes were found at 1.060 g/ml or higher densities. A discontinuous gradient of FH with 2 layers of 1.060 and 1.077 g/ml of density respectively was selected to obtain trypomastigotes-free white blood cells from blood samples. The functional capacity of lymphocytes recovered from the lower interface, where no parasites were found, was assessed. The response to phytohaemagglutinin of these high density lymphocytes was as good as of total lymphocytes, suggesting that low density lymphocytes are not necessary for proliferative responses. It is postulated that high density lymphoid populations, free from T. cruzi forms, may be used to study the presence of T cell-mediated immune response in Chagas' disease patients.     

An improved method for purification of lymphocytes

A method that combines glass-bead column filtration, Ficoll-Hypaque gradient separation, discontinuous sucrose gradient, and drastic reduction of cell transfers is described. The procedure gives a high yield of pure human lymphocytes from small amounts of blood, good preservation of B cell/T cell ratio, and sufficient material for subsequent biochemical studies.     

四种常用的人中性粒细胞分离方法的比较

目的：比较Percoll非连续密度梯度离心法、Ficoll-Hypaque 密度梯度离心法、裂解红细胞法和Dextran作用下红细胞自然沉降法四种常用的人中性粒细胞分离方法。方法：取健康人外周静脉血，分别采用以上四种方法进行中性粒细胞分离，对其细胞纯度、回收率、存活率进行比较。结果：Percoll非连续密度梯度离心法与Ficoll-Hypaque密度梯度离心法分离得到的细胞纯度均大于90%，两者间比较无统计学差异（P＞0.05）；裂解红细胞法和Dextran作用下红细胞自然沉降法分离得到的细胞纯度略低于Percoll非连续密度梯度离心法(P＜0.01)与Ficoll-Hypaque密度梯度离心法（P＜0.05）。Dextran作用下红细胞自然沉降法的回收率低于Percoll非连续密度梯度离心法（P＜0.01）、Ficoll-Hypaque密度梯度离心法(P＜0.01)和裂解红细胞法（P＜0.05）；Percoll非连续密度梯度离心法回收的中性粒细胞存活率明显高于Ficoll-Hypaque密度梯度离心法（P＜0.05），裂解红细胞法(P＜0.01）和Dextran作用下红细胞自然沉降法（P＜0.01）。结论：Percoll非连续密度梯度离心法分离中性粒细胞，纯化程度好，回收率高，是一种简单、高效的中性粒细胞分离方法，适于临床和科研中广泛应用。     

Rapid visual detection of microorganisms in blood culture.

We describe a method and apparatus for rapid visual detection of microorganisms in blood cultures. In the 30-min procedure, a lysing reagent for the preferential digestion of blood cells and a concentration device which causes 1 ml of lysed specimen to pass through a portion (3 mm2) of a membrane filter (pore size, 0.6 micron) were used. After the material remaining on the filter was Gram stained, the filter was mounted and examined microscopically. The ability to detect microorganisms in blood cultures was determined by spiking seven common blood pathogens into blood cultures prepared from the blood of healthy volunteers. Microorganism concentration in the cultures ranged from 1 to 1,000/ml. All of 34 cultures with at least 100 CFU/ml were detected, 34 of 64 cultures with less than 100 CFU/ml were detected, and 41 of 42 negative controls were correctly reported as negative.     

Countercurrent centrifugal elutriation in a table-top centrifuge

An elutriation system has been developed which fits into a small table-top centrifuge. The new CS1 rotor functioned as accurately as the JE-6 rotor, commercially available from Beckman Instruments. However, fewer cells were required for the separation experiments. Leukocytes obtained from 25 ml whole blood by Ficoll-Hypaque gradient centrifugation were sufficient for separating lymphocytes from monocytes and for purifying granulocytes. Furthermore, when lymphocytes were harvested from cell cultures 40 ml of a suspension of 1.2 x 10(6) cells/ml were enough to yield an enriched preparation of antibody producing cells.     

巨噬细胞Ia抗原表达和抗原提呈功能调控的研究——Ⅵ.人单核细胞表面HLA-Ⅱ类抗原和IL-2R与其抗原提呈功能的关系

取健康青壮年男性献血员的新鲜外周静脉血,肝素抗凝,经密度梯度离心获得单个核细胞。又经粘附法和尼龙毛柱分离法分别获得高纯度的单核细胞和T淋巴细胞。用单克隆抗体分别阻断单核细胞表面的 HLA-DR、HLA-DQ、HLA-DP 抗原,经洗涤后,将单核细胞与PPD共同温育24小时,观察单核细胞向T淋巴细胞提呈PPD的能力。另一组先将单核细胞与 PPD作用24小时,然后用单克隆抗体阻断单核细胞表面的 IL-2R1小时,再观察单核细胞的抗原提呈功能。结果表明,分别阻断单核细胞表面的 HLA-DR、DQ、DP 和IL-2R 后,其抗原提呈能力均显著减弱。     

Isolation and Characterization of Rat Trophoblast Cells

ABSTRACT: Previous immunohistochemical studies of the rat placenta using specific alloantisera and/or monoclonal antibodies showed that the basal zone trophoblasts stained for Pa and A^a class I major histocompatibility complex (MHC) antigens and for the human SP1-related antigen. In an effort to isolate the basal zone trophoblast cells from the rat placenta, we used these markers to assess the degree of purification of the cells separated by density gradient centrifugation using either Ficoll-Hypaque or Percoll as the gradient medium. The cells were put either on the top or at the bottom of discontinuous density gradients in the range of 1.005-1.10 or/ml. The cell separation profiles for the two media were different. With Percoll, most of the trophoblast cells (80–95%) were collected at the density gradients 1.04/1.06 and 1.06/1.08, whereas with Ficoll-Hypaque, these gradients separated only a small fraction (4–23%) of the trophoblast cells, and most of them pelleted at the bottom of the tube. The trophoblast cells separated by Ficoll-Hypaque, however, showed fewer contaminant cells than those separated by the Percoll gradients.     

Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide.

Human neutrophils were isolated from peripheral blood by four methods: 1) Ficoll-Hypaque gradients and erythrocyte lysis, 2) plasma-Percoll gradients, 3) a "lipopolysaccharide (LPS)-free" method yielding 85% neutrophils, and 4) by centrifugation of cells prepared by Method 3 through a plasma-Percoll gradient to produce pure neutrophils. The use of the Ficoll-Hypaque method resulted in spontaneous change of cell shape, enhanced formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated release of superoxide anion, increased release of lysosomal enzymes upon subsequent FMLP stimulation, and reduced chemotactic responsiveness, by comparison with the other methods. These effects were not due to erythrocyte lysis by NH4C1 but were reproduced by exposure of neutrophils prepared by the "LPS-free" method or the use of plasma-Percoll gradients to 10-100 ng/ml LPS. Neutrophil change of shape and stimulated O-2 production were particularly sensitive markers of these effects. The effects of trace concentrations of LPS in the modulation of neutrophil function may have relevance to the pathophysiology of endotoxemia and its resultant tissue injury.     

Improved separation of rhesus monkey lymphocytes with Percoll

The total yield and the proportions of Rhesus monkey peripheral blood mononuclear cells (PBMC) with detectable T4, T8, T11 and Ia markers that were purified by density gradient centrifugation through solutions of polyvinyl pyrrolidone (Percoll) were significantly greater than those found in PBMC suspensions obtained by using Ficoll-Hypaque. The percentages of recovery of PBMC and of cells expressing the above markers were lowest among PBMC isolated with a Ficoll-Hypaque mixture of density 1.077 and highest among PBMC obtained by using a solution of Percoll of density 1.077. Percoll of density 1.077 was also better than Ficoll-Hypaque in that it yielded PBMC with the lowest levels of erythrocyte (less than 1%) and granulocyte (less than 1%) contamination. The reduced levels of PBMC with detectable markers appeared to result from an effect of either Ficoll or Hypaque on the lymphocytes rather than from loss of lymphocyte subpopulations since higher proportions of cells bearing T4, T8, T11 and Ia were found when PBMC were isolated with a solution of Percoll which yielded a percentage of PBMC as low as that recovered with Ficoll-Hypaque. Altered marker expression by cells obtained with Ficoll-Hypaque was not seen with similarly prepared human PBMC.     

Superior leukocyte separation with a discontinuous one-step Ficoll-Hypaque gradient for the isolation of human neutrophils

A discontinuous gradient system composed of two commercially available Ficoll-Hypaque mixtures (Mono-Poly resolving medium (MPRM) and Histopaque 1.077) is described for the purification of mononuclear and polymorphonuclear leukocytes from human blood. Like the original one-step Hypaque-Ficoll procedure (Ferrante and Thong, 1978), a single centrifugation at 500 × g for 30 min resulted in the formation of two distinct leukocyte fractions. In contrast to MPRM alone, the discontinuous system (MPRM-HP) was capable of resolving leukocyte fractions from blood volumes as small as 1 ml with excellent purity and yield. MPRM-HP was also compatible with a wider range of anticoagulants and permitted fractionation of specimens resistant to MPRM.     

Rapid single-step method for purification of polymorphonuclear leucocytes from blood of patients with rheumatoid arthritis.

Previous studies have shown that blood samples from healthy subjects and patients with a variety of different diseases separate into distinct mononuclear leucocyte (MNL) and polymorphonuclear leucocyte (PMNL) bands when centrifuged on a Ficoll-Hypaque medium of density 1.114 g/ml. Results from the present study showed that blood samples from patients with rheumatoid arthritis (RA) failed to separate into discrete cellular fractions under the same conditions. In particular, the PMNL were not recoverable because the erythrocytes only partially sedimented. However, following modification of the separation medium involving (a) reduction in the final Ficoll concentration from 8% to 7%, and (b) an alteration in the ratio of meglumine Hypaque:sodium Hypaque from 5.7:2.8 to 2.0:7.5, a clear band of PMNL of high purity could be obtained with blood samples from patients with RA. The modification of this previously described, simple, brief, one-step, density gradient centrifugation method achieved separation of peripheral blood PMNL required for studies of leucocyte function in patients with RA.     

Characterization of canine neutrophil granules.

The purpose of this study was to isolate distinct populations of canine neutrophil granules and to compare them with neutrophil granules from other species. Size, shape, density, and content of canine neutrophil granules were determined. Neutrophils obtained by Ficoll-Hypaque sedimentation were homogenized, and granule populations were separated by isopycnic centrifugation on a linear sucrose gradient (rho, 1.14 to 1.22 g/ml). The most dense granule population (rho, 1.197 g/ml) contained all of the myeloperoxidase, beta-glucuronidase, and elastase, more than half of the acid beta-glycerophosphatase, and most of the lysozyme. The population with intermediate density (rho, 1.179 g/ml) contained lactoferrin, vitamin B12-binding protein, and the remainder of the acid beta-glycerophosphatase and lysozyme. The least dense granule population did not contain a major peak of any of the enzymes or binding proteins tested but was distinguished by density and morphology. The size and shape of the granules were determined from scanning electron micrographs and assessment of shape was aided by transmission electron micrographs. By these methods three populations of canine neutrophil granules were characterized and named: myeloperoxidase granules, vitamin B12-binding protein granules, and low-density granules.     

Leucocyte behaviour in controlled ischaemia of the calves.

Whole blood filterability and leucocyte behaviour (number, activation, and subfraction filterability rates) were monitored at the earliest stage of peripheral ischaemia in 18 patients with stage II peripheral occlusive arterial disease (PAOD) and 20 matched controls. A model of controlled ischaemia, using exercise to stress leg circulation, was set up and blood samples were taken before exercise, at the onset of calf pain, and at recovery from peak exercise. Leucocytes were counted, separated into their subfractions on a Ficoll-Hypaque density gradient and by adhesion to Petri dishes, and filtered in buffer (like the whole blood suspensions) through 5 microns pore diameter Nucleopore filters. Unfractionated white cells, separated under gravity, with pseudopodia or cytoplasmic irregularities were regarded as activated. The whole blood filterability rate was significantly increased at the onset of calf pain and was associated with significant increases in the number of leucocytes and in the filterability rate of the monocyte subfraction, the latter persisting throughout the recovery period. No significant changes were observed in the other variables monitored, showing that impairments in white cell rheology may be associated with ischaemia.     

Simultaneous preparation of mononuclear and polymorphonuclear leucocytes from horse blood on ficoll-hypaque medium

Results presented show that highly purified populations of mononuclear (MN) and polymorphonuclear (PMN) leucocytes can be obtained from horse blood by a procedure similar to that previously described for the separation of these leucocytes from human blood. This involved centrifugation of horse blood on a Ficoll-Hypaque medium with a density of 1.095 g/ml. The procedure required approximately 1 h for completion and resulted in the simultaneous preparation of MN (>98% purity) and PMN (>96% purity) leucocytes. Cell viability exceeded 95% and cells retained immunological functions.     

Normal levels of lymphotoxin secretion by freshly isolated and refrigerated human peripheral blood lymphocytes

Lymphocytes obtained by Ficoll-Hypaque density gradient centrifugation of leukocytes from 40 blood bank donors were incubated at 10(6) cells/ml for 24 h with 5 micrograms phytohemagglutinin/ml serum-free RPMI 1640 medium and secreted 34 +/- 6 U lymphotoxin/ml and 52 +/- 29 U interferon/ml. Addition of 5% fetal bovine serum (FBS) or acid-citrate-dextrose treated autologous plasma (AP) augmented lymphotoxin secretion 2-fold and interferon secretion 6-8-fold. Lymphotoxin alone was secreted in 39% of serum free cultures. Both lymphotoxin and interferon were secreted in the presence of FBS or AP. Lymphocytes refrigerated at 4 degrees C for 18 h secreted upon serum-free culture similar levels of lymphotoxin as fresh cells but cultures secreting lymphotoxin alone increased from 39 to 77%. Lymphotoxin secretion by refrigerated lymphocytes was similar in the presence or absence of FBS or AP. Interferon secretion by refrigerated lymphocytes in the presence of FBS or AP, however, increased 30-40-fold approaching levels produced by freshly isolated cells and indicating that the modulatory effects of refrigeration, FBS, and AP affected interferon not lymphotoxin secretion. The levels of lymphotoxin and interferon secreted by lymphocyte populations producing both lymphokines, furthermore, varied independently of one another providing additional evidence for the dissociability of their secretion yet possible dependence of interferon upon lymphotoxin secretion. The ability to reliably quantitate lymphotoxin secretion using refrigerated lymphocytes permits transport of lymphocytes to laboratories performing quantitative bioassay of the lymphokine. This should facilitate further evaluation of the presence and role of lymphotoxin in immunopathological and other disease states.     

Improved method for recovery of peritonitis-causing microorganisms from peritoneal dialysate.

The efficacy of recovery of peritonitis-causing microorganisms from peritoneal dialysate fluid by using the Septi-Chek blood culture system (Roche Diagnostics, Div. Hoffmann-La Roche Inc., Nutley, N.J.) was compared with those of other conventional techniques, such as the 20-ml culture and the filtration methods. The recovery of microorganisms by using the Septi-Chek system was found to be as effective as the filtration of 250 ml of dialysate that used a modified Millipore filtration technique (Millipore Corp., Bedford, Mass.). Both methods were found to be superior to the 20-ml culture method. We suggest using the Septi-Chek method as the standard protocol for the culture of dialysate as a relatively inexpensive and labor-saving recovery technique.     

Induction of neovascularization by activated human monocytes

Neovascularization, the process of new blood vessel growth, is an important feature of many pathologic and physiologic processes. Monocytes were isolated from citrated blood buffy coat of healthy adult human donors on Ficoll-Hypaque gradients. Mononuclear cells from these gradients were fractionated on discontinuous Percoll gradients; monocyte-enriched fractions were isolated and assessed for angiogenic activity in rat corneas. Freshly isolated monocytes as well as monocytes cultured for 20 hr on fibronectin-coated collagen gels failed to stimulate neovascularization. In contrast, adherent monocytes activated with concanavalin A (25 micrograms/ml) or endotoxin (5 micrograms/ml) for 20 hr were found to be potently angiogenic. We conclude that peripheral blood monocytes must be activated to acquire the ability to induce new blood vessel growth, a process central to inflammation, wound healing, and tumor development.