Lack of subclass selection during density purification of human lymphocytes.

The percentage of acid alpha-naphthyl acetate esterase (ANAE) marker-carrying lymphocytes (T-lymphocytes) was determined directly from the blood, after density purification of lymphocytes over Ficoll-Isopaque and removal of monocytes by iron powder plus magnet. The effect of Ficoll-Isopaque centrifugation was insignificant; neither did the iron powder plus magnet procedure increase the relative proportion of the T cells in these preparations. We therefore conclude that in optimal conditions there is no subclass-specific enrichment of T or B lymphocytes during the purification procedure.     

Three-step isolation of human blood monocytes using discontinuous density gradients of Percoll

A three step method for the purification of normal human blood monocytes is described. The procedure consists of a combination of dextran sedimentation, Ficoll-Isopaque (F-I) centrifugation and isopycnic centrifugation on discontinuous gradients of Percoll. No selective loss of monocytes was observed after the first step, and after F-I centrifugation mononuclear cells (MNC) were obtained, of which 20 +/- 6% were monocytes. The MNC were further separated on hyper-osmotic and iso-osmotic discontinuous density gradients of Percoll. The best purification of monocytes occurred on hyper-osmotic density gradients and the density interface between 1.074 and 1.066 g/ml yielded 85 +/- 7% monocytes, 13 +/- 7% lymphocytes and 1 +/- 1% granulocytes. 77 +/- 16% of the monocytes obtained after F-I centrifugation, were recovered in this interface. The purified monocytes were viable and retained their capacity to mature into macrophages. The whole procedure takes about 5 h, is reproducible and can be applied to small and large volumes (500 ml) of blood.     

A two-step procedure for obtaining normal peripheral blood T-lymphocytes using continuous equilibrium density gradient centrifugation on percoll

Equilibrium centrifugation of either peripheral blood mononuclear cells or of pure lymphocytes (obtained by carbonyl iron or glass bead adherence removal of monocytes) on a continuous density gradient of Percoll yielded lymphocyte fractions containing between 92 and 99% T lymphocytes as shown by sheep red blood cell rosetting. B lymphocytes with surface immunoglobulin were found in the regions of low density (1.03-1.065 g/ml) and T lymphocytes in the regions of higher density (1.06-1.08 g/ml). TM lymphocytes with their characteristic positive 'dot' pattern of staining for non-specific esterase were also found mainly in regions of high density. It was concluded that Percoll continuous equilibrium density gradient centrifugation can be used to obtain T lymphocytes in high yield, with high viability and without metabolic changes which may occur after contact with sheep red blood cells. The esterase staining suggested that there was also some separation of T lymphocyte subsets.     

T-lymphocyte and B-lymphocyte dichotomy in anuran amphibians: I. T-lymphocyte proportions, distribution and ontogeny, as measured by E-rosetting, nylon wool adherence, postmetamorphic thymectomy, and non-specific esterase staining

We used sheep erythrocyte (SRBC) E-rosetting, nylon wool fractionation thymectomy and nonspecific esterase staining to assess T-lymphocyte characteristics in Rana pipiens during various developmental stages. T-lymphocytes appear in the spleens of premetamorphic tadpoles. We found significant levels of lymphocytes classifiable as T-cells in the peripheral blood of adults, but fewer numbers of T-cells in the thymus, jugular bodies, spleen, pronephros, mesonephros, liver and bone marrow. In addition, thymectomized Xenopus laevis showed a sharp decrease in T-cells as evidenced by E-rosetting, ANAE stain and nylon wool fractionation. The presence of T-lymphocytes in anuran amphibians and the existence of a receptor for SRBC on a population of these cells, suggest conservation of T-cells during evolution.     

Efficient separation of human T lymphocytes from venous blood using PVP-coated colloidal silica particles (percoll)

In a comparative study, human peripheral T lymphocytes were separated as E rosettes by density centrifugation through various gradient media. Sheep red blood cells (SRBC) were removed by dissociation of the E rosettes at 37 degrees C with subsequent centrifugation on a similar density gradient prewarmed to 37 degrees C. In particular, gradients made of Ficoll Urovison were compared with Percoll gradients with regard to both separation steps. Using Percoll gradients, a maximal T cell recovery of 75% was obtained, whereas Ficoll separation yielded only 46%. T lymphocytes separated with Percoll exhibited equal viability compared to Ficoll isolated cells and consisted of 98% EAET-RFC. No inhibition of cellular function by Percoll treatment was detected, whereas Ficoll treatment led to an impaired mitogenic response. An inherent mitogenicity of Percoll was not observed. The method described results in considerably shortened centrifugation times due to the low viscosity of the Percoll medium and simultaneously seems to be less harmful to the rather fragile rosettes. Reproducibility was found to depend on careful control of density and osmolarity of the Percoll medium.     

Separation of human peripheral blood monocytes on continuous density gradients of polyvinylpyrrolidone-coated silica gel (Percoll)

A standardized, reproducible two-step method for separation of human peripheral blood monocytes on continuous Percoll gradients has been developed. The first step involves separation of mononuclear cell on Percoll of density 1.075 g/ml and the second step separation of monocytes from lymphocytes on a continuous Percoll gradient with a starting density of 1.075 g/ml for the formation of the gradient. The average yield during a 10 month period of daily routine use has been 74 +/- 17% (mean +/- 1 S.D.), and the average purity 63 +/- 10%. Ninety to 95% of the monocytes are viable after separation as judged from trypan blue exclusion and by ingestion of latex particles and sensitized sheep erythrocytes. The separation takes about 3 h and the total number of monocytes obtained from 40 ml of blood is in the range of 10-15 x 106. The procedure has been reliable with 3-4% separation failures, mainly due to bacterial or fungal growth in Percoll suspension or media. The contaminating cells are exclusively lymphocytes, predominantly T-lymphocytes (90-95%), when citrate is used as anticoagulant. Heparin can not be used as anticoagulant, as there appears to be a dose-dependent formation of thrombocyte aggregates which contaminate the monocytes, and result in poor separation.     

Classification of normal and malignant lymphatic cells using acid phosphatase and acid esterase

Summary The usefulness of cytochemical tests (APh and ANAE) to replace or to supplement membrane markers in subclassification of normal and malignant lymphatic cells was investigated. Material: normal lymphocytes subfractionated by rosetting and centrifugation, and in M. Hodgkin and CLL; lymphoblastoid cell lines; malignant lymphatic cells in different types of lymphatic leukemias. In normal human blood, T-lymphocytes are marked by a distinct dot-like ANAE-reactivity which is somewhat less pronounced in the small (11%) subgroup of Fc-IgG-receptor positive T-lymphocytes; B-lymphocytes are negative or finely granular positive. Lymphoblastoid cell lines of B- and of T-type are ANAE- and APh-positive. In some lymphatic malignancies, a characteristic pattern of activity of APh or of ANAE may support the diagnosis. The value of ANAE-cytochemistry is highly estimated for the quantitative determination of the percentage of normal T-lymphocytes in lymphatic leukemias, immunological disorders, and during immunosuppressive therapy.

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Abbreviations ANAE acid -naphthyl acetate esterase - APh acid phosphatase - (c-)ALL (common-type) acute lymphatic leukemia - CLL chronic lymphatic leukemia - E(AET)-lymphocytes lymphocytes spontaneously forming rosettes with (AET-treated) SRBC - Fc-IgG-lymphocytes lymphocytes forming rosettes with IgG-sensitized ORBC - MGG May-Grünwald-Giemsa staining of blood films - Non-E-lymphocytes lymphocytes not forming rosettes with SRBC (from interphase of Ficoll-separation) - ORBC ox red blood cells - SRBC sheep red blood cells - (T)-ALL (T-cell-type) acute lymphatic leukemia - T-IgG-lymphocytes E(AET)-lymphocytes forming rosettes with IgG-sensitized ORBC - T-non-IgG-lymphocytes E(AET)-lymphocytes non-rosetting with IgG-sensitized ORBC     

Responses of human large granular lymphocytes and classical T-cells in culture to allogeneic cells, lectins, and soluble antigen

Subpopulations of human peripheral blood mononuclear lymphocytes, after depletion of B-cells and monocytes (by sequential adherence to plastic and nylon wool columns) were separated by Percoll density gradient centrifugation and tested for their ability to proliferate in response to various mitogens, recall antigens, and alloantigens, and to develop cytolytic reactivity in vitro. The low-density fraction [mostly large-granular lymphocytes (LGL)] contained greater than 95% of the total cytotoxic activity of unfractionated nonadherent lymphocytes, against the natural killer (NK)-susceptible K562 target cell, and against other NK-susceptible targets, whereas the high-density lymphocyte fraction (mostly classical T-lymphocytes) demonstrated little or no cytolytic activity against these targets. Conversely, cytotoxic alloreactivity against the lymphoblasts of the donor used for stimulation developed only in the cultures of high density cells. Autologous cytotoxic reactivity, against autologous phytohemagglutinin (PHA)-stimulated lymphoblasts, was not restricted to either subset but developed by both LGL as well as high-density lymphocytes. LGL and high-density T-lymphocytes demonstrated significant proliferative responses to lectins [PHA, concanavalin A (Con A), pokeweed mitogen], and the responses of the LGL were similar in magnitude to those of peripheral blood mononuclear cells or of high-density T-cells. In contrast, only T-lymphocytes responded to the specific recall antigen, purified protein derivative (PPD). These results indicate that LGL are capable of proliferative responses to various lectins, but have no detectable specific memory responses to a soluble antigen. In addition, a different subset of lymphoid cells was responsible for the development of NK-like and specific alloreactivity. Therefore, NK cells and T-cells, although sharing proliferative responses to mitogens, exhibit different function regarding cytotoxic effectors.     

Human large granular lymphocytes contain an esterase activity usually considered as specific for the myeloid series

A cytochemical marker such as alpha-naphthyl acetate esterase (ANAE) has been found useful for the morphological identification of the subset of T lymphocytes having receptors for Fcμ (T_M cells). ANAE reaction on T_M cells gives a typical pattern of one to four positive spots, whereas this pattern is not found on T cells with receptors for Fcγ (T_G cells). ANAE is abundant in monocytes but not detectable in granulocytes. Herein another type of esterase activity, naphthol-AS-D chloroacetate esterase (NCAE), is described; it is known to be abundant in granulocytes and was found to give a specific pattern of reactivity with the subpopulation of large granular lymphocytes (LGL). This pattern of fine granular staining was observed not only on LGL present in the T_G cell subpopulation but also in LGL present in the non-T, non-B cells. Fractions of peripheral blood mononuclear cells which were ènriched up to 80% in LGL by Percoll discontinuous density gradient gave a similar percentage of specific NCAE pattern. In addition, among the different fractions from Percoll gradient, there was a good correlation (r = 0.94) between the number of NCAE-positive cells and the natural killer activity against the natural killer susceptible K562 target cells. It will be important to determine whether or not this enzymatic activity plays a role in the cytotoxic activities of LGL.     

用Percoll法、Ficoll法及常规法制备的效应细胞杀伤功能的比较

本文用Percoll配成的不连续密度梯度(Percoll法)对C57BL、C_(3H)和CBA品系小鼠的脾细胞进行了分离,并用不同密度梯度层分离所得的细胞对~3H-TdR或~(51)Cr标记的YAC-1靶细胞进行杀伤功能检测.发现以50～60％、60～70％二个密度梯度层之间的细胞杀伤活性最高,poly I:C可以增强这种活性.并且在同一效靶比例下,这种效应细胞对YAC-1靶细胞的杀伤活性均高于用Ficoll-Isopague分离的脾细胞(Ficoll法)及常规制备的脾细胞(常规法),前者与后二者所获数据间呈显著性差异(p<0.01)。     

Functional Relationship of Human T Lymphocytes, Monocytes and Interleukins

The different requirements of human T lymphocytes of different densities for accessory cells and helper factors (Interleukin 1 (Il-1) and Interleukin 2 (Il-2) ) in the response to phytohaemagglutinin (PHA) were investigated. Human T lymphocytes were subfractionated by discontinuous density gradient centrifugation. The various T-cell subsets were stimulated by PHA to form colonies in an agar micro-culture in the presence or absence of additional adherent cells or crude preparations of Il-1 or Il-2. The results show that the higher the density of the fractionated T lymphocytes and the lower the number of cells cultured, the greater is the number of adherent cells or the amount of helper factors required for the stimulation of colony-forming T lymphocytes. The results are consistent with the assumption that monocytes provide positive modulating activity during mitogenic stimulation of colony-forming T lymphocytes. The number of monocytes necessary for exerting an optimal modulating activity depends on the number of T cells cultured and the density of the T-cell fraction. This may reflect a distinct susceptibility of T cells of different densities to monocyte-mediated helper effects.     

Human Peripheral Blood Null Lymphocytes Stimulated by Staphylococcus aureus Cowan I Produce Atypical Acid-Labile Interferon in Vitro

Peripheral blood mononuclear leucocytes (PBLs) stimulated in vitro by heat-killed formaldehyde-fixed Staphylococcus aureus Cowan I (SACoI) produced acid-labile alpha interferon (IFN-alpha) and, to various extents, also IFN-gamma. The IFN producers resided in nylon wool-nonadherent cells, and monocytes suppressed SACoI-induced IFN responses. Further separation of nonadherent PBLs in accordance with expression of surface antigenic markers was performed with a 'panning' technique. The SACoI induced production of IFN in cells that carried neither surface immunoglobulins nor OKT3-defined antigens. These cells were also characterized as OKM1- and OKT10-negative. In contrast, cells with natural killer (NK) activity against K562 erythroleukaemia cells were located in both OKM1- and OKT10-positive and -negative cells. At centrifugation on Percoll density gradients, cells with NK activity and IFN response against SACoI were recovered from light gradient fractions that contained mainly large granular lymphocytes (LGL). Furthermore, the IFN producers were enriched by removal of sheep erythrocyte-rosetting T cells from the Percoll fractions. These SACoI-induced IFN-producing PBLs are LGL but lack certain antigens that are frequently found on NK cells.     

人外周血NK细胞纯化、扩增及克隆化的初步探索

分别采用Panning法、补体裂解法、绵羊红细胞花环法、Percoll不连续密度梯度离心法对NK细胞进行纯化并以流式细胞仪检测其纯度和计数探索NK细胞的体外扩增条件 ,将纯化NK细胞经有限稀释 ,在饲养细胞、IL 2、PHA及LCM等培养条件下进行克隆化培养并进行鉴定。研究表明NK细胞的纯度由纯化前 10 %左右提高到 30 %～ 70 %不等 ,以Percoll不连续密度梯度离心法所获纯度最高 ,约为 70 %。NK细胞的体外扩增在含有IL 2、PHA及条件培养基最显著。每 96孔板可获 4～ 16个CD3 CD5 6 +NK细胞克隆 ,每 96个克隆的细胞数最多可达 2 35× 10 5。     

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.     

Separation of cytotoxic lymphocytes against Marek's disease lymphoblastoid cell lines on Percoll gradients

A two-step method has been developed for the separation of lymphocytes cytotoxic for allogeneic Marek's disease lymphoblastoid cell lines. Ficoll-Paque-purified spleen cell suspensions were separated according to their densities on continuous gradients of Percoll. Pooled fractions were tested for specific release in a 4-hour chromium release assay. Cytotoxic activity was mainly detected in the fractions with a density of 1.060 to 1.070 g/ml. Levels of specific release were increased using those fractions compared to Ficoll-Paque-purified cells. A discontinuous Percoll gradient was developed based on the density range of cytotoxic cells. Continuous and discontinuous Percoll gradients were equally effective for the preparation of cytotoxic cells.     

Large granular lymphocytes: Morphological studies

Large granular lymphocytes from normal human blood were enriched by centrifugation on discontinuous Percoll density gradients. Their capacity for natural killing, but not for phagocytosis of yeast cells, was demonstrated. Large granular lymphocytes are characterized in electron microscopy by their fine structure, especially by typical granules and by inclusions of tubular structures in a parallel array. Their lymphocyte nature is supported by activity of acid-α-naphthyl acetate esterase and by the absence of myelo-peroxidase (POX) and of macrophage POX. The Fcγ receptor of their cell membrane is marked by soluble POX-anti-POX-complexes; labeled parts of their membrane are not incorporated into the cytoplasm as in monocytes.     

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。     

Simple and cost-effective isolation of monocytes from buffy coats

We have optimized the procedure of monocyte isolation on a Percoll density gradient. The new procedure consists of three steps: (1) the isolation of MNC on a Ficoll density gradient; (2) the separation of monocytes from lymphocytes on a high-density hyper-osmotic Percoll density gradient; and (3) the separation of monocytes from platelets and dead cells on a low-density iso-osmotic Percoll density gradient. The procedure is simple and cost-effective. Monocyte purity and recovery are both about 75% and platelet contamination is low. The isolated monocytes retain their capacity to differentiate into dendritic cells in vitro.     

Cell size monitored counterflow centrifugation of human bone marrow resulting in clonogenic cell fractions substantially depleted of small lymphocytes

Human bone marrow cells were fractionated by physical methods in order to obtain cell fractions enriched in clonogenic cells and devoid of immunocompetent lymphocytes. The bulk of the erythrocytes was removed by isopycnic gradient centrifugation on Ficoll-Isopaque (d = 1.085 g/ml) and the majority of mature granulocytes on Percoll (d = 1.070 g/ml). The nucleated cells were separated into fractions by counterflow centrifugation. Continuous monitoring of the effluent of the elutriator by a light scatter device improved the reproducibility of the separation profiles. Progenitor cells did not form a single distinct peak and the maximal enrichment factor was 8.5. Lymphocytes were eliminated almost completely from the progenitor cell rich fraction (both CFU-GM and BFU-E). Physical elimination of lymphocytes from human bone marrow may offer an alternative approach to the prevention of graft-versus-host disease in allogeneic bone marrow transplantation.     

Bovine peripheral blood leukocyte subpopulation dynamics following a primary bovine herpesvirus-1 infection

Population dynamics of bovine peripheral blood leukocyte subpopulations were quantitated following a primary bovine herpesvirus-1 (BHV-1) infection. Percoll isolated peripheral blood mononuclear cell (PBMC) subpopulations were analyzed using flow cytometry (FC) and cytochemical stains. Between days two to eight post-infection (PI) there was a significant decrease in the percentage of T-cells and nonT/nonB cells which was accompanied by an increased percentage of B-cells and monocytes. These percentages were extrapolated to the number of Percoll isolated PBMC during this period. A decrease in the T-cell population was the primary cause of the observed lymphopenia and a relative increase in the percentage of B-cells. The increased percentage of monocytes was caused by an increased number of circulating monocytes. These monocytes were characterized by an increase in Fc receptor expression, a decrease in plastic and Sephadex-G10 adherence and no apparent change in the level of class II MHC antigen (Ia) expression. Serum cortisol was significantly elevated on day 2 PI and may have been responsible for both the reduction in circulating T-cells and a decrease in the in vitro viability of peripheral blood lymphocytes. The percentage of Ia positive PBMC was increased significantly on day 4 PI. However, on days 4 and 6 PI the summated percentages of monocytes and B-cells (total Ia expressing population) exceeded significantly the actual percentage of Ia positive cells. This apparent suppression of Ia expression did not coincide with the elevated serum prostaglandin E2 concentrations on days 8 and 10 PI.