A flow cytometric method for determination of absolute counts of myeloid precursor dendritic cells in peripheral blood

Circulating precursor dendritic cells (pDCs) constitute a rare population in peripheral blood. They have a typical immunophenotypic profile, yet, they cannot be identified by pDC-specific immunophenotypic markers and therefore, their accurate and absolute enumeration poses a challenge. Here, we report a method for the evaluation of absolute counts of myeloid pDC in minimally manipulated blood samples on a flow cytometer as a single platform. Three-color flow cytometry was done to identify myeloid pDC as CD33+ HLA-DR+ CD14/CD16(dim/negative) cells in commercially available TruCount trade mark tubes that contain a defined number of brightly fluorescent polystyrene beads. The normal range in peripheral blood of 41 healthy adults, as determined by this single-platform method, was 17.0+/-5.7 x 10(6)/l, or 0.64+/-0.23% of mononuclear cells (MNCs). In parallel experiments, we have compared our procedure with two published 'dual-platform' methods that derive the absolute pDC count from a relative number obtained by flow cytometry, and from absolute counts obtained from a haematological analyser. Regression analysis showed an excellent correlation between results obtained with our single-platform protocol and these double-platform procedures (R2 > or = 0.90). However, the values obtained by the single-platform method were significantly higher than those obtained by the dual-platform methods. The higher myeloid pDC numbers in this single-platform procedure are likely due to reduced cell loss in this 'lyse-no-wash' protocol compared with the other methods which include density gradient separation and centrifugation steps. The intra- and interassay variability were 4.4% (range, 2.04-8.96%) and 5.8% (range, 2.59-9.65%), respectively. Thus, the single-platform method described here allows accurate, rapid and simple measurement of circulating blood myeloid pDC and is suitable for routine enumeration of circulating myeloid pDC.     

Effect of SIVmac infection on plasmacytoid and CD1c+ myeloid dendritic cells in cynomolgus macaques

Dendritic cells (DCs) are known to be essential for the induction and regulation of immune responses. Non-human primates are essential in biomedical research and contribute to our understanding of the involvement of DCs in human infectious diseases. However, no direct single-platform method for quantifying DC precursors has yet been optimized in macaques to give accurate absolute blood counts of these rare-event cell populations in the blood. We adapted a rapid whole-blood assay for the absolute quantification of DCs in cynomolgous macaques by four-colour flow cytometry, using a single-platform assay compatible with human blood. Cynomolgus macaque plasmacytoid DCs (pDCs) and CD1c⁺ myeloid DCs (CD1c⁺ mDCs) were quantified in the blood of 34 healthy macaques and the results obtained were compared with those for blood samples from 11 healthy humans. In addition, circulating absolute numbers of pDCs were quantified in cynomolgus macaques chronically infected with SIVmac. During infection, pDC counts decreased whereas circulating CD1c⁺ mDC counts increased. Information regarding absolute pDC and mDC counts in non-human primates may improve our understanding of the role of these cells in SIV/HIV infection and in other infectious diseases.     

The impact of erythrocyte lysing procedures on the recovery of hematopoietic progenitor cells in flow cytometric analysis

Since preanalytic lysing of erythrocytes remains critical in flow cytometry, we investigated the influence of four lysing procedures on the quantification of leukocyte and CD34+ cells in hematopoietic cell transplants (HCTs). Samples were derived from stem cell-enriched mobilized whole blood collected by apheresis (unselected) and immunologically purified stem cell products (selected) and were measured using the dual-platform (2-PF) method with two flow cytometric systems. Additionally, cells were measured by a volume-based technique (single platform [1-PF]). Results were identical in the 2-PF mode (unselected HCTs, r = 0.998; selected HCTs, r = 0.999). In comparison with the 2-PF results, the single-platform (1-PF) measurements revealed a mean decrease of 59.5% for CD34+ cells (50.8% for CD45+ cells) in unselected HCTs and a mean decrease of 52% for CD34+ cells (49.8% for CD45+ cells) in selected HCTs. In order to check the accuracy of cell quantification using the 1-PF method, leukocyte reference values from hematology counter results were compared with flow cytometric (1-PF)-counted nucleated cells. That analysis revealed good congruency, with r = 0.998 for unselected HCTs and r = 0.999 for selected HCTs. In conclusion, all lysing procedures that we used induced substantial loss of leukocytes and CD34+ cells. As demonstrated by the high accuracy of the 1-PF technique, all erythrocyte lysing procedures caused significant cell loss, which led to inconsistent counting of CD34+ cells in nonvolumetric flow cytometric (2-PF) protocols.     

Change from dual- to single-platform reporting of CD4/CD8 values: experience from a small district general hospital laboratory

Accurate and reliable CD4 and CD8 counts are essential for monitoring HIV disease progression or successful therapy. CD4 and CD8 counts can be determined on a flow cytometer by either single- or dual-platform technology. Dual-platform technology uses a haematology analyser to obtain a total white cell count and lymphocyte absolute count. CD4 and CD8 absolute values are then calculated from the CD4 and CD8 percentage positive results obtained from the flow cytometer. Single-platform technology uses latex beads of a predefined concentration, which are added to the blood sample immediately before flow cytometric analysis, thereby removing the need to use an additional analyser. Recent recommendations propose that single-platform technology should be the gold standard for CD4 measurement because it offers better inter-laboratory coefficients of variation (CVs). Before changing to single-platform technology in our department, CD4 and CD8 absolute counts, determined on 20 healthy volunteers, were used to establish new normal ranges for single-platform technology (Coulter epics XL), permitting absolute value data for dual-platform and single-platform technologies to be compared. Data obtained with single-platform technology was significantly higher for both CD4 and CD8 (P=0.001 and P=0.003, respectively). For CD4, mean single-platform value was 0.993 x 10(9)/L (+SD = 0.510-1.376) and dual-platform value was 0.920 x 10(9)/L (+SD = 0.500-1.340). For CD8, single-platform value was 0.483 x 10(9)/L, (+SD = 0.207-0.756) and dual-platform value was 0.457 x 10(9)/L (+SD = 0.222-0.692). Thus, the differences between dual- and single-platform absolute CD4 and CD8 results were small (8% and 6%, respectively) but significant. It is important, therefore, that clinicians closely monitoring CD4 and CD8 values and are informed of any laboratory changes.     

Loss of CD34(+) hematopoietic progenitor cells due to washing can be reduced by the use of fixative-free erythrocyte lysing reagents

Current protocols for sample preparation before flow cytometric enumeration of CD34(+) hematopoietic progenitor cells (HPC) include both lyse-non-wash and lyse and wash methods. Erythrocyte lysis without washing is the method of choice when absolute cell counts are to be assessed, whilst a washing step is recommended for immunological subtyping of CD34(+) cells in order to reduce background fluorescence. Here, we analyzed the effect of the interaction between type of erythrocyte lysis reagent and washing on the outcomes of (i) CD34(+) cell enumeration and (ii) expression of CD38 by CD34(+) cells in a single-platform, whole-blood staining assay [Gratama, J.W., Keeney, M., Sutherland, D.R., 1999. Enumeration of CD34(+) hematopoietic stem cell and progenitor cells. Curr. Protocols Cytometry 6(4), 1-22.]. We studied seven commercially available lysing reagents (five containing fixative and two fixative-free) using 12 samples from cord blood (n=4), mobilized peripheral blood (n=4) and apheresis products (n=4). Using the lyse and wash technique, significant reductions of absolute and relative numbers of CD34(+) cells, as well as in the numbers of lymphocytes and leukocytes, were observed on samples that had been lysed using fixative-containing buffers as compared to the lyse-no-wash technique. Cell losses due to washing could be significantly reduced when samples were lysed using fixative-free buffers. 'Postfixation' using PBS+1% paraformaldehyde of samples that had been lysed using fixative-free buffers and then washed did not result in additional loss of CD34(+) cells or other cell types. Finally, washing unfixed samples led to a slight decrease of CD38 monoclonal antibody bound to CD34(+) cells as compared to samples that had been fixed during erythrocyte lysis. These results indicate that fixation renders (CD34(+)) cells sticky and leads to their loss from the cell suspension upon centrifugation and resuspension. We conclude that all seven lysing reagents can be used with confidence in a lyse-no-wash technique, but that only fixative-free lysing reagents should be used when a washing step is considered necessary.     

Establishment of adult peripheral blood lymphocyte subset reference range for an Asian population by single-platform flow cytometry: influence of age, sex, and race and comparison with other published studies

We established a normal reference range for peripheral blood lymphocyte subsets in a multiracial adult population by using single-platform flow cytometry. Further analysis of our cohort showed that the CD8+-cell counts decrease with age, there is a gender difference in NK cell percentages and counts, and there are significant differences in the CD3+-, CD4+-, and CD19+-cell counts between Indians and other racial groups. Overall, our results are significantly different from other published data. This difference further stresses the need for different populations to establish their own reference ranges as these may have important implications for the management of patients with human immunodeficiency virus and AIDS. The use of single-platform flow cytometry will eliminate some of the variability between different study centers, making studies more comparable. This platform should be used for future studies into the effects of age, sex, and race on lymphocyte subsets.     

A reliable and inexpensive EasyCD4 assay for monitoring HIV-infected individuals in resource-limited settings

Serial measurements of absolute CD4+ T-lymphocyte counts are required to initiate and gauge response to therapy and monitor disease progression. Hence, there is an urgent need to evaluate the accuracy and validity of low-cost CD4+ T-cell count assays. Tripotassium EDTA blood specimens from HIV-infected individuals were studied using a novel flow cytometric assay (EasyCD4 assay; Guava Technologies, Hayward, CA) in comparison with standard flow cytometry (FACSCount; Becton Dickinson Immunocytometry Systems, San Jose, CA). The sensitivity, specificity value by EasyCD4 assay in enumerating absolute CD4+ T-cell counts of less than 200 cells/microL were 95% and 100%, respectively. Bland-Altman analysis showed close agreement, with the EasyCD4 assay yielding CD4+ T-cell counts a mean difference of -26 cells/microL (95% confidence interval, -96 to 44 cells/microL) higher than by flow cytometry. Our data suggest that EasyCD4 assay could be a useful alternative assay to conventional flow cytometry, may be appropriate for use in resource-limited settings.     

Quantification of mucosal mononuclear cells in tissues with a fluorescent bead-based polychromatic flow cytometry assay

Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P<0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.     

广州市健康儿童和成人淋巴细胞亚群数量的对比分析

目的比较儿童和成人淋巴细胞亚群百分率和绝对数量的异同,更好地为本地区临床诊断和治疗提供参考。方法采用双色流式细胞术分析健康儿童和成人外周血T淋巴细胞亚群(CD3+细胞、CD3+CD4+细胞、CD3+CD8+细胞)、B淋巴细胞(CD19+)、CD3-CD56+NK细胞(自然杀伤细胞)的数量。结果儿童组总淋巴细胞(百分率和绝对数)、CD19+淋巴细胞(百分率和绝对数)、CD3+淋巴细胞百分率、CD3+CD4+淋巴细胞百分率、CD3+CD8+淋巴细胞绝对数、CD3-CD56+细胞绝对数、CD4+/CD8+细胞比值与成人组比较差异均有统计学意义;CD3+CD8+淋巴细胞百分率、CD3-CD56+细胞百分率、CD3+淋巴细胞绝对数和CD3+CD4+淋巴细胞绝对数与成人组比较差异无统计学意义。儿童组CD3+CD8+淋巴细胞、CD19+淋巴细胞、CD3-CD56+细胞的百分率和绝对数均高于成人组,CD3+淋巴细胞(百分率和绝对值)、CD3+CD4+淋巴细胞(百分率和绝对值)和CD4+/CD8+细胞比值低于成人组。相同与不同性别组内均有多个指标存在显著差异。结论淋巴细胞亚群的分布受年龄、性别因素的影响,淋巴细胞亚群绝对数与百分率随年龄的变化不总是保持一致的。用于血液性和免疫性疾病诊断时,采用淋巴细胞亚群绝对数作为参考指标优于百分比率。     

Immunohematological reference values for healthy adults in Burkina Faso

Reference ranges for peripheral blood lymphocyte subsets were generated for 186 healthy adults in Burkina Faso using single-platform flow cytometry. CD4(+) T-cell counts ranged from 631 to 1,696 cells microl(-1); they were lower in males (n = 97) than in females (n = 89), whereas natural killer cell counts were higher.     

Absolute CD4 T-cell counting in resource-poor settings: direct volumetric measurements versus bead-based clinical flow cytometry instruments

Flow cytometry is an accurate but expensive method to determine absolute CD4 cell counts. We compared different methods to measure absolute CD4 counts in blood samples from HIV-infected and uninfected subjects using a research/clinical flow cytometer (FACScan); a dedicated clinical instrument (FACSCount); and a volumetric, mobile, open-system flow cytometer equipped with 3 fluorescence and 2 light scatter detectors (Cyflow SL blue). The FACScan and Cyflow were used as single-platform instruments, but they differ in running cost, which is a central factor for resource-poor settings. Direct volumetric and bead-based CD4 measurements on the Cyflow were compared with 2 bead-based single-platform CD4 measurements on the FACSCount and on FACScan (TruCount) in "Le Dantec" Hospital, Dakar, Senegal, using whole blood samples from 102 HIV+ and 28 HIV- subjects. The agreement between the various measurement methods was evaluated by Bland-Altman analysis. Volumetric CD4 measurements on the Cyflow using a no-lyse-no-wash (NLNW) procedure and a lyse-no-wash (LNW) procedure correlated well with each other (R2 = 0.98) and with CD4 measurements on the FACSCount (R2 = 0.97) and FACScan (R2 = 0.97), respectively. Red blood cell lysis had no negative effect on the accuracy of absolute CD4 counting on the Cyflow. An excellent correlation was observed between bead-based CD4 measurements on the Cyflow and CD4 measurements on the FACSCount (R2 = 0.99) and FACScan (R2 = 0.99). Rigid internal and external quality control monitoring and adequate training of technicians were considered essential to generate accurate volumetric CD4 measurements on the Cyflow.     

不同预处理移植后树突状细胞早期重建及与移植物中CD34+细胞相关性分析

目的：比较不同预处理异基因造血干细胞移植（allo-HSCT）后早期树突状细胞（DCs）亚群重建情况，以及移植物中CD34^＋细胞是否影响移植后早期DCs亚群重建。方法：采用三色流式细胞仪动态检测不同预处理移植后早期外周血树突状细胞亚群DC1、DC2水平。结果：移植后早期清髓性移植患者体内DCs亚群数量非常低，常规移植组移植后14天与半相合移植组相比，DC1、DC2均无统计学意义（P〉0．05）。非清髓性移植组（NST）DC1、DC2高于清髓性移植组，两者相比具有统计学意义（P〈0．05）。在30天和60天，所有组DC1、DC2略有波动，但是幅度不大。以输入的CD34^＋细胞数平均分为三组，三组患者DC1、DC2移植后14、30和60天均无统计学意义（P〉0．05）。结论：NST后患者早期DCs重建较清髓性干细胞移植患者早，而常规移植和半相合移植早期DCs重建较慢，二者无差别。移植物中的CD34^＋细胞不影响移植后早期DCs亚群重建。     

Natural killer (NK) cell subsets and NK cell cytotoxicity in women with histories of recurrent spontaneous abortions

PROBLEM: Natural Killer (NK) cell measurement and NK cytotoxicity are two measurements for assessing the cellular immune response. Both of the techniques have been reported to be prognostic for women with recurrent spontaneous abortion (RSA), We evaluated the two methods to determine the relationship of the two assays. Because both methods portend to evaluate the same process, the previous clinical data suggested that the methods evaluate the same phenomena. We undertook these studies to determine whether simple NK cell counts may be sufficient in the evaluation of NK activity in RSA. METHOD OF STUDY: The NK cell cytotoxicity at effector-to-target ratios of 50:1 and 25:1 was determined using a flow cytometric NK cell cytotoxicity assay. These values were then correlated with the percentages and absolute counts of three peripheral blood NK cell subsets. RESULTS: The data indicate that the flow cytometric assay is reproducible and precise and can be successfully used to evaluate patient samples. Linear regression analysis indicated a lack of correlation between peripheral blood NK cell cytotoxicity and percentages or absolute counts of ***CD56+CD16+, CD56+CD16 — or CD3+CD56+ lymphocyte subsets (range of correlation coefficients, 0.1–0.3). CONCLUSIONS: NK cell cytotoxicity and peripheral blood NK cell values measure different aspects of NK cells and do not correlate. These data indicate that simple enumeration of NK cells may not be sufficient in the evaluation of NK cells in RSA.     

An immunomagnetic single-platform image cytometer for cell enumeration based on antibody specificity

Simplification of cell enumeration technologies is necessary, especially for resource-poor countries, where reliable and affordable enumeration systems are greatly needed. In this paper, an immunomagnetic single-platform image cytometer (SP ICM) for cell enumeration based on antibody specificity is reported. A chamber/magnet assembly was designed such that the immunomagnetically labeled, acridine orange-stained cells in a blood sample moved to the surface of the chamber, where a fluorescent image was captured and analyzed for cell enumeration. The system was evaluated by applying one kind of antibody to count leukocytes and one kind for each leukocyte subpopulation: CD45 for leukocytes, CD3 for T lymphocytes, and CD19 for B lymphocytes. Excellent precision and linearity were achieved. Moreover, these cell counts, each from blood specimens of 42 to 52 randomly selected patients, were compared with those obtained by SP (TruCount) and dual-platform (DP) flow cytometry (FCM) technologies. The cell counts obtained by our system were in between those obtained from the TruCount and DP FCM methods; and good correlations were achieved (R > or = 0.95). For CD4(+) counts, as we expected, the cell count by our system was significantly higher than the CD4(+) T-lymphocyte counts obtained by SP and DP FCM methods. Immunophenotyping of the immunomagnetically selected CD4(+) cells showed that, besides CD4(+) T lymphocytes, a proportion of the CD4(+) dim monocytes was also selected. Our system is a simple immunomagnetic SP ICM, which can potentially be used for enumeration of CD3(+) CD4(+) T lymphocytes in resource-poor countries if an additional CD3 immunofluorescent label is applied.     

自体外周血造血干细胞移植后造血重建中CD226分子在NK细胞上表达的变化

本文应用双重免疫荧光染色和流式细胞术分析,观察自体外周血造血干细胞移植患者造血重建中,CD226分子在CD56bright和CD56dimNK细胞亚群表达的变化.结果显示,在自体外周血造血干细胞移植造血重建中,于干细胞回输第12天,CD56 NK细胞占外周血淋巴细胞百分率为26.6%,其中CD56bright亚群,占CD56 NK细胞87.3%;CD56 CD226 细胞占CD56 NK细胞92.1%,CD56brightCD226 细胞占CD56 CD226 细胞89.9%.在自体外周血造血干细胞移植造血重建中,CD56bright亚群是NK细胞造血重建最早出现并占优势的一个亚群, CD226分子可能作为一种分化标记主要表达在CD56bright亚群上.     

In vivo expansion of the circulating stem cell pool

Stem cell trafficking between extravascular marrow sites and circulating blood is an essential part of the blood stem cell transplantation technology. Recombinant human G-CSF (rHuG-CSF) is widely used for stem cell peripheralization alone or together with chemopriming mobilizing early and pluripotent CD34+ cell subsets. New cytokine/chemokine mobilization regimens are under investigation such as combined rHuG-CSF and rHu thrombopoietin, rHuG-CSF and interleukin 3, rHuG-CSF and rHu stem cell factor, rHuG-CSF and Flt-3 ligand, human macrophage inflammatory protein, interleukin 1, and interleukin 8. Modifying the adherence of CD34+ cells to extracellular matrix molecules is a new mechanism by which hematopoietic progenitor cells are released into the circulating blood. Blocking the alpha4beta1 integrin receptor on CD34+ progenitor cells by using monoclonal antibodies specific for the heterodimeric complex alpha4beta1 has been shown to further increase the circulating stem cell concentration when given following rHuG-CSF priming. The current clinical research is primarily focused on improving stem cell mobilization efficiency in heavily pretreated and poorly mobilizing patients, and to decrease adverse effects of cytokine treatment.     

Studies of the site and distribution of CD34+ cells in idiopathic myelofibrosis

The frequency and distribution of CD34+ cells in the bone marrow (BM) of patients with idiopathic myelofibrosis (IM) were determined using an immunohistochemical technique. The percentage and absolute number of circulating CD34+ cells were enumerated. Patients with IM exhibited a continuum of number of BM CD34+ cells ranging from 1 to 85 per 5 mm(2). The frequency of BM CD34+ cells was inversely related to the number of circulating CD34+ cells. The BM biopsy specimens obtained from 4 patients with IM who underwent allogeneic stem cell transplantation were examined sequentially. Quantitative measurement revealed that the reticulin fiber volume was progressively reduced after allogeneic stem cell transplantation. All 4 patients had normocellular marrow with normal numbers of BM CD34+ cells after transplantation. These findings suggest that the BM fibrosis and abnormal hematopoietic stem cell distribution in patients with IM is a consequence of the progeny of a malignant hematopoietic stem cell clone.     

Long-term immune recovery of patients undergoing allogeneic stem cell transplantation: a comparison with their respective sibling donors.

We have addressed whether patients' immune system status after allogeneic stem cell transplantation, assessed more than 1 year after the procedure, recovers normal function as compared with that of their respective donors. An additional aim was to compare the status of the immune system between patients receiving reduced-intensity conditioning regimens and those undergoing myeloablative transplantations. For this purpose, we analyzed not only the different subsets of peripheral blood (PB) lymphocytes, but also circulating dendritic cell (DC) subpopulations, together with cytokine production by PB T cells, in a series of 38 patients undergoing allogeneic stem cell transplantation. We compared these patients with their respective HLA-matched donors by performing a simultaneous patient/donor paired study. Complete bone marrow chimerism status and normal PB cell counts were demonstrated in all recipients. The most relevant numeric differences found between patients and donors were related to the distribution of the distinct subsets of PB DCs (CD16+ DCs were increased, whereas myeloid and plasmacytoid DC subsets were decreased in the patient group). This was associated with an increased number of B cells, an inverted CD4/CD8 T-cell ratio, and a decrease in CD4+/CD8+ double-positive T cells in the patient group. In addition, a predominance of a T-helper 1 pattern of cytokine production (interferon gamma and tumor necrosis factor alpha) with decreased secretion of T-helper 2-associated cytokines (interleukin 5 and interleukin 10) was also observed at the single-cell level. No significant differences were found in any of the parameters analyzed between patients receiving reduced-intensity conditioning regimens and those undergoing myeloablative transplantations.     

A high capacity in vitro assay for measuring the cytoadherence of Plasmodium falciparum-infected erythrocytes

A novel flow cytometric technique was developed to determine the absolute numbers of leukocytes of specific phenotypes in whole blood from two lines of inbred chickens (line 7(2) and line 6(1)). This single step method is rapid, accurate, repeatable, can be used in the presence of nucleated erythrocytes and addresses the problems encountered when electronically counting the numbers of leukocytes in specific subpopulations in the blood of non-mammalian species. It is superior to previous methods in that (1) peripheral blood leukocytes (PBL) do not need to be separated by density gradient centrifugation, (2) erythrocyte lysis is not necessary and (3) absolute numbers of specific phenotypes of cells are determined directly. A standard volume of diluted whole blood was added to a standard number of fluorescent beads before incubation with fluorescently-conjugated monoclonal antibodies recognising specific PBL surface antigens. Samples were analysed by flow cytometry and electronic gates were set to count a standard number of beads and the concomitant fluorescently-labelled cells. Absolute numbers of B, CD4+ and CD8+ PBL were determined. Since the bead fluorescence is constant, it was also possible to measure relative MHC class I expression using fluorescence intensity. In both lines of chickens absolute numbers of all of the phenotypes of PBL measured increased with age. Although line 7(2) chickens had greater numbers of B, CD4+, and CD8+ PBL than line 6(1) chickens, there was no significant difference in the CD4+:CD8+ PBL ratios, the T:B PBL ratios or relative MHC class I expression between the two lines. Relative MHC class I expression increased with age in both lines.     

肾移植后外周血CD4+及CD8+T细胞亚群计数的临床意义

背景：临床上常以流式细胞检测受者外周血CD4、CD8细胞比值来揭示与排斥或感染相关的关系。 目的：探讨肾移植后排斥或感染时外周血CD4⁺及CD8⁺T细胞(简称CD4和CD8细胞)亚群计数的变化和意义。 方法：应用流式细胞仪检测肾移植121例受者CD4、CD8细胞数进行检测。根据入院病情将患者分为移植后正常组、急性排斥组、肺部感染组进行观察。 结果与结论：移植后正常患者和急性排斥患者相比,CD4、CD8细胞数差异均无显著性意义(P > 0.05)。肾移植后肺部感染患者CD4、CD8细胞数则均显著低于移植后正常组(P < 0.001)。当感染控制、症状改善时,CD4、CD8细胞数显著升高 (P < 0.001)。说明肾移植后CD4和CD8细胞计数可以作为免疫状态的参考,其对于感染的参考价值大于排斥,动态观察分析有助于指导治疗。