Quantitative study of KI-67 antibody staining in non-Hodgkin's lymphomas using image analysis.

In sections from 32 B malignant lymphomas (ML), the total KI-67 stained area was compared to the number of KI-67 positive cells in order to demonstrate the reliability of using image analysis to quantify the proliferative activity. The total KI-67 area percentage correlated highly with the number of KI-67 positive cellular profiles (r = .93). Significant differences were found between low- and high-grade ML according to the Kiel classification (mean values +/- SD, respectively, of 7.7 +/- 3.81% and 16.6 +/- 6.23%), and between low-, or intermediate- and high-grade ML only, according to the International Working Formulation. Within the Working Formulation, the statistical analysis grouped the diffuse large cell subtype of intermediate grade with the immunoblastic high-grade subtype. A wide range of KI-67 area percentage values was noted, particularly in follicular ML; for these follicular ML, considering follicular areas only, values were comparable to high-grade ML (14.8 +/- 6.60%). In conclusion, the KI-67 area percentage is a reliable alternative method to manual cell counting, and image analysis allows quicker measurements appropriate to large and strictly lymphomatous areas, using a greater number of cells than in manual cell counting.     

Assessment of cell proliferation by Ki-67 staining and flow cytometry in fine needle aspirates (FNAs) of reactive lymphadenitis and non-Hodgkin's lymphomas

This study was undertaken to assess cell proliferation in FNAs from a series of 57 non-Hodgkin's lymphomas (NHL) and 11 cases of reactive lymphadenitis using Ki-67 staining and flow cytometry (FCM). The results were compared and correlated to the cytomorphological subgrouping according to Kiel classification. The mean percentages of Ki-67 positivity were 16.6% and 61.1% for low and high grade lymphomas, respectively (P < 0.001). The mean S-phase fraction (SPF) determined by FCM was 4.61% for low grade and 12.9% for high grade lymphomas (P < 0.001). The figures for Ki-67 positivity and S-phase fraction in reactive lymphadenitis were 16.8% and 40%, respectively. We observed a strong correlation in low grade lymphomas between Ki-67 and SPF. A good correlation was also found in reactive lymphadenitis. In high grade lymphomas, however, with highly scattered Ki-67 and S-phase values, this correlation was lost. In some cases this discrepancy can be explained by a rich admixture of non-neoplastic, non-proliferating cells in aspirates from diploid tumours. In addition, the existence of a minor aneuploid tumour cell population of high proliferation such as that in Ki-1 lymphomas will not be accurately analysed by FCM but is easily assessed by Ki-67 staining. However, the main reason seems to be a high variability between the fraction of cells in S-phase and the total number of cells in G1, S and G2 in individual tumours.     

Lymphocyte subsets and viral load in patients with HIV-associated non-Hodgkin's lymphoma treated with anti-CD20 monoclonal antibody and chemotherapy

The anti-CD20 monoclonal antibody Rituximab is a novel antitumor agent used in association with chemotherapy (CT) for the treatment of high-grade/intermediate non-Hodgkin's lymphomas (NHL) in HIV-negative populations. This therapeutic combination is currently also being explored in HIV-positive patients with NHL (HIV-NHL). The objective of our study was to determine CD4 and CD8T cell counts, HIV plasma viremia and proviral load in patients with CD20-positive HIV-NHL treated with Rituximab plus CT and highly active antiretroviral therapy (HAART). We studied eight patients with HIV-NHL treated by anti-CD20 and CT before, after three, and after six cycles of therapy; CD4, CD8 and CD19 lymphocyte subsets were measured by monoclonal antibodies and flow cytometry. HIV plasma viremia was determined by the b-DNA assay, and proviral load by a quantitative competitive PCR. CD4T cell counts remained stable after three cycles of therapy, while a significant reduction of this subset was present at the end of therapy. HIV plasma viremia was significantly reduced after the third cycle, but returned to pretreatment levels at the end of therapy; we also observed individual fluctuations of proviral load during therapy, this marker being increased in two out of three patients at the end of therapy. These observations suggest that Rituximab plus CT accelerated the rate of CD4 depletion and of HIV replication in the peripheral blood of HIV-NHL patients and that HAART may be able to delay these effects. Received: 1 December 2000 / Accepted: 8 February 2001     

Analysis of apoptosis by propidium iodide staining and flow cytometry

Since its introduction, the propidium iodide (PI) flow cytometric assay has been widely used for the evaluation of apoptosis in different experimental models. It is based on the principle that apoptotic cells, among other typical features, are characterized by DNA fragmentation and, consequently, loss of nuclear DNA content. Use of a fluorochrome, such as PI, that is capable of binding and labeling DNA makes it possible to obtain a rapid (the protocol can be completed in about 2 h) and precise evaluation of cellular DNA content by flow cytometric analysis, and subsequent identification of hypodiploid cells. The original protocol enhanced the capacity for a rapid, quantitative measure of cell apoptosis. For this reason, since its publication, the PI assay has been widely used, as demonstrated by the large number of citations of the original paper and/or the continuous use of the method in many laboratories.     

MHC class II determinants on peripheral blood monocytes from newly diagnosed IDDM patients.

The expression of MHC class II determinants (HLA-DR, HLA-DP and Ia7) on peripheral blood monocytes (OKM1+ cells) was studied in 20 children with newly diagnosed IDDM. Monocytes of 10 children with IDDM and familial predisposition showed a statistically significant increase of HLA-DR expression when compared to control group (10 healthy children). There were no significant differences concerning Ia7 expression. HLA-DP expression was similar in all studied groups.     

One-round determination of seven leukocyte subsets in rhesus macaque blood by flow cytometry

BACKGROUND: Rhesus macaques are frequently used in biomedical research as experimental models for studying infectious diseases and for preclinical vaccination trials. The infection of these monkeys with simian immunodeficiency viruses (SIV) or simian-human immunodeficiency viruses (SHIV) reproduces the clinical and immunological characteristics of human infection by human immunodeficiency virus (HIV). Evolution of the immune response in the infected animals is generally analyzed by determining the lymphocyte subsets on blood samples using flow cytometry but requiring multiple, blood consuming, determinations. METHODS: Cell subsets present in whole-blood samples were labeled with a combination of anti-human monoclonal antibodies to CD2, CD20, CD4, CD8, and CD14 coupled to FITC or PE and analyzed by flow cytometry. RESULTS: In one round, we obtained the precise determination of macaque blood cell composition by flow cytometry. Monocytes, granulocytes, eosinophils, B lymphocytes, helper, and cytotoxic T lymphocytes were distinguished. Results obtained correlated strongly with those obtained with conventional blood cell differential systems and with separate staining of lymphocytes. The analysis of blood from healthy rhesus macaques and SHIV-infected animals demonstrated the accuracy of the determination even in very pathological situations such as macaques with simian AIDS. CONCLUSIONS: Our method allows fast determination of the blood cell composition and will be particularly useful to evaluate the cell subset evolution of macaques involved in large-scale experimental trials.     

BCG in the immunotherapy of Hodgkin's disease and non-Hodgkin's lymphomas

Summary Patients with bad prognosis malignant lymphomas were treated by a combination of radiotherapy and polychemotherapy. After complete remission they were randomized: one group was treated by BCG in dermic scarification, the other one was not treated. The results do not ascertain BCG efficiency but justify the intensive reductive treatment.Immuno-oncology week, Paris, June 27, 1975     

Depressed cytotoxic T-cell responses in previously treated Hodgkin's and non-Hodgkin's lymphoma patients

Summary Peripheral blood lymphocytes from 62 previously treated Hodgkin's and non-Hodgkin's lymphoma patients were tested for their ability to generate cytotoxic T lymphocytes in response to stimulation with allogeneic cells in mixed leukocyte culture. In most patients, including some in long-term unmaintained remission, extremely low cytotoxic responses were generated. To test whether these patients have circulating cells that suppress autologous lymphocytes from responding to alloantigens, patients' responding cells were passaged over columns of sepharose beads conjugated with histamine-rabbit serum albumin (Hist-RSA). This procedure has been shown to remove mouse suppressor cells and Concanavalin A(ConA)-induced human suppressor cells. Passage of patients' cells, prior to allogeneic stimulation, over columns of sepharose beads conjugated with Hist-RSA but not over control RSA columns, resulted in the isolation of lymphocytes that generated increased cytotoxic responses to alloantigens in 18 of 22 patients with initially low cytotoxic responses. These results suggest that the impaired ability of treated Hodgkin's and non-Hodgkin's lymphoma patients' lymphocytes to differentiate into cytotoxic T lymphocytes is at least in part due to the presence of circulating suppressor cells that bear histamine receptors.Scholar of the Leukemia Society of America     

Analysis of peripheral blood lymphocyte subsets and prognosis in patients with septic shock

The purpose of the study is to study the relationship between peripheral blood lymphocyte subset proportion and prognosis in patients with septic shock. Fifty‐two patients with septic shock, admitted to the intensive care unit between March 2007 and December 2010, were enrolled in this study. Peripheral blood lymphocyte subset proportions were measured using flow cytometry. The percentage of CD3⁺CD4⁺ T lymphocytes and CD19⁺ lymphocytes, CD4⁺/CD8⁺ T cell ratio were substantially lower in patients with septic shock compared to the control group (P < 0.01). The percentage of CD3⁺CD8⁺ T lymphocytes did not differ significantly between the two groups (P > 0.05). The percentage of CD16⁺CD56⁺ lymphocytes was higher in patients with septic shock than in the control group (P < 0.01). Compared with the survivor group, the percentage of CD3⁺CD4⁺ T lymphocytes and CD19⁺ lymphocytes, CD4⁺/CD8⁺ T cell ratio were clearly lower in the non‐survivor group (P < 0.01). There was no difference in the percentage of CD3⁺CD8⁺ T lymphocytes between the non‐survivor and survivor groups (P > 0.05). The percentage of CD16⁺CD56⁺ lymphocytes was higher in the non‐survivor group than in the survivor group (P < 0.05). The total maximum SOFA score and the delta SOFA score were much higher in the non‐survivor group than in the survivor group (P < 0.01). Immune imbalance occurs in patients with septic shock. Peripheral blood lymphocyte subset proportion and SOFA scores can be used to assess the treatment and prognosis of septic shock.     

Analysis of malaria parasite-infected blood by flow cytometry

The use of flow cytometry in the quantitative analysis of blood from mice infected with Plasmodium vinckei has been studied. Several fluorescent dyes responsive to cell membrane potential were screened and one dye, 3,3'-dimethyloxacarbocyanine (DiOC1(3) ), was chosen for further study. Mature red blood cells (mRBC), immature RBC (imRBC), and parasitized RBC (pRBC) could be recognized and counted in the flow cytometer. When infected blood was separated on a Percoll gradient and fractions analyzed by flow cytometry using DiOC1(3), distinct populations of pRBC were recognized, the frequency of which varied with density. These subpopulations could not be correlated with distinct morphologic stages but varied with the size or age of the growing parasite. Methods combining the use of DiOC1(3) with a DNA specific-dye, Hoechst 33342, are discussed as an approach to more complete analysis of the blood of malaria-infected animals.     

小细胞肺癌患者外周血淋巴细胞亚群分析

目的 探究化疗对小细胞肺癌(small cell lung cancer,SCLC)患者免疫功能的影响。 方法 选择2013年1月到2018年12月我院收治的95例小细胞肺癌患者为研究对象。患者第一周期、第二周期化疗前采用流式细胞术检测患者外周血淋巴细胞亚群水平,分别按照不同疗效及不同化疗方案对患者外周血淋巴细胞亚群进行比较。 结果 (1)化疗后,95例患者CD3+、CD4+、CD8+细胞平均值增加,CD19+、γδT细胞平均值减少,差异均有统计学意义(均P+、CD8+细胞平均值增加,CD19+细胞减少,差异有统计学意义(均P0.05)。(3)依托泊苷联合顺铂(EP)方案组化疗后患者CD3+、CD8+细胞平均值增多,CD19+细胞减少,差异均有统计学意义(均P0.05)。 结论 化疗可以调节小细胞肺癌患者的免疫功能,增强细胞免疫,降低体液免疫,其中EC方案对患者细胞免疫的增强作用较为显著。     

Analysis of chromosomes from human peripheral lymphocytes by flow cytometry

A method of preparation and flow cytometric analysis of chromosomes from human peripheral lymphocytes is described. The procedure allows a resolution coefficient of variation better than 3% using propidium iodide staining and a commercially available flow cytometer.     

Continuous measurement and analysis of staining kinetics by flow cytometry

The measurement of color development with time in cells following the start of a staining reaction is of interest in a number of biological systems. These include the subsets of peripheral white blood cells after acridine orange staining, the uptake by cells and nuclei of fluorescent agents, especially antitumor drugs, and measurement of intracellular enzyme kinetics using fluorogenic or absorbing substrates. The present work describes a simple computer program for analyzing flow cytometric (FCM) data versus time, including both the population kinetics of color development and the variability of staining speed within one population of cells. A single-channel absorption measurement in flow (Technicon Hemalog D) was used to record peroxidase kinetics in peripheral blood cells. Every 5 s, a 64-channel absorption histogram was recorded, up to a maximum of 64 histograms. The data were then analyzed by a computer program which searched for the peak channel of each histogram. A least-squares fit was computed for these maxima. The asymmetries of the 64 absorption histograms were compared to see if there was more than one population present with different time constants. Although developed for enzyme kinetic measurements, this program may have wider usefulness in any measurements of time-dependent phenomena by FCM.     

Quantitation of human anti-mouse antibody in serum by flow cytometry.

Clinical investigations utilizing murine monoclonal antibodies require techniques for the detection of the human anti-mouse antibody (HAMA) response in patient serum. We report here a flow cytometric assay for the quantitation of HAMA. Commercially available beads conjugated with goat anti-mouse antibody provide a solid phase matrix for a triple bridge immunoassay. The measurement of fluorescein labeled antibodies by flow cytometry allows accurate quantitation of the HAMA. The assay will detect antibody levels of approximately 1.0 ng/ml. Antibody recovery in serum samples with known amounts of antibody added was greater than 90% at levels greater than or equal to 10 micrograms/ml. Serum samples obtained from 41 patients prior to and after single or multidose infusions of KS1/4-Desacetyl-vinblastine were analyzed. These results were compared with HAMA titers previously determined by ELISA. With few exceptions, patients with low titers as determined by ELISA demonstrated low HAMA potencies by flow cytometry and those with highest titers the highest potencies. Patients with no detectable HAMA by ELISA were also negative by flow analysis. The results of our studies demonstrate that HAMA levels can be accurately and quantitatively determined by flow cytometry.     

Flow cytometry,with double staining with Nile red and anti-CD3 antibody,to detect phospholipidosis in peripheral blood lymphocytes of rats treated with amiodarone

A flow cytometry method, to monitor peripheral lymphocytes phospholipidosis, has been set up using a single staining with Nile red and double staining with Nile red and anti-CD3 monoclonal antibody. Blood has been collected from rats treated with amiodarone (phospholipidogenic antiarrhythmic drug). By flow cytometer, it is possible to detect phospholipids, using Nile red, a probe for intracellular lipids staining, changing its fluorescence on the stained lipid basis. CD3 antigen has been selected to focus on T cells, to evaluate whether these cells are the target of phospholipidosis amiodarone-dependent. In the study A, Sprague–Dawley rats were treated with three different doses (75, 150, and 300 mg kg⁻¹ day⁻¹) of amiodarone or vehicle alone, for 14 days, followed by 14 days of recovery: Data obtained show that by flow cytometry, with Nile red alone, it is possible to detect a dose- and time-related response of phospholipidosis-positive lymphocytes; a partial recovery is also assessed. In the study B, Sprague–Dawley rats were treated with a single dose (300 mg kg⁻¹ day⁻¹) of amiodarone, for 14 days: Data obtained show that animals treated with amiodarone have a significant increase of phospholipidosis-positive lymphocytes (p = 0.008), in particular of CD3⁺ cells (p = 0.0056). Transmission electron microscopy analysis confirmed data obtained by flow cytometry. This work shows that flow cytometry with Nile red could be a good tool to monitor ex vivo phospholipidosis in lymphocyte cells of animals treated with amiodarone: The phospholipidogenic effect is more evident focusing on CD3⁺ T lymphocytes, thus suggesting that these cells are probably the target of phospholipidosis.     

Cerebellar degenerative changes in patients with acute leukaemias and non-Hodgkin's lymphomas.

In patients with neoplasms located outside the central nervous system can appear degenerative changes within nervous tissue. Neuropathological investigations have been done on 101 cases with acute Non-Lymphoblastic Leukaemias (ANLL) and Non-Hodgkin's Lymphomas (NHL). Cerebellar degenerative changes especially granular layer rarefaction or atrophy appear more frequently in ANLL than in NHL.     

Increased peripheral blood gamma delta T-cells in patients with lymphoid neoplasia: A diagnostic dilemma in flow cytometry

We have observed increased numbers of non-neoplastic gammadelta-T-cells in the peripheral blood of a series of patients with non-Hodgkin's lymphoma not of gammadelta-T-cell origin. The majority of normal gammadelta-T-cells are negative for surface CD4 and CD8 and a subpopulation does not express CD5, two immunophenotypic findings strongly suggestive of neoplasia in alpha beta T-cells. In addition, they express cytotoxic T-cell/Natural killer cell antigens. In this study, up to 22% of PBLs were CD4 and CD8 negative gammadelta-T-cells and up to 33% PBLs were CD5 negative gammadelta-T-cells. In addition, as high as 42% of PBLS were gammadelta-T-cells expressing cytotoxic T-cell/Natural killer cell antigens, suggestive of a large granular lymphoproliferative disorder. Failure to recognize that these are normal gammadelta-T-cells could lead to the erroneous diagnosis of peripheral blood involvement with a T-cell neoplasm, especially in the setting of a history of non-Hodgkin's lymphoma. Cytometry (Comm. Clin. Cytometry) 38:280-285, 1999. Published 1999 Wiley-Liss, Inc.     

Detection of adenocarcinoma in peritoneal washings by staining with monoclonal antibody B72.3

Peritoneal washings are routinely performed in the staging evaluation of carcinomas of the ovary and in "second-look" explorations; major problems in the evaluation of these specimens continue to be the distinction between atypical mesothelium and adenocarcinoma and the identification in an otherwise inflammatory specimen of rare cells of adenocarcinoma, which may be undetected by the most trained individual. Monoclonal antibody (MAb) B72.3, reactive with an oncofetal, tumor-associated glycoprotein (termed TAG-72; MW greater than 1000 kd) expressed in a variety of epithelial malignancies but not generally expressed in benign or malignant mesothelium, was reacted with sections from the paraffin-embedded cell blocks of 185 peritoneal washings from 180 patients with extant cancer or a prior history of malignancy. One hundred four of the washings were initially interpreted as atypical mesothelium, with no evidence of malignancy; when reacted with MAb B72.3, 6 of these specimens demonstrated groups of metastatic adenocarcinoma cells not appreciated by the usual cytologic criteria. Of the 81 washings interpreted as showing cells of adenocarcinoma, 73 demonstrated expression of TAG-72 from both gynecologic and nongynecologic malignancies. In the remaining 49 cases without an associated malignant process, MAb B72.3 did not stain atypical mesothelium, but did react, however, with benign endometrial cells and müllerian inclusions in two cases. MAb B72.3 may be used as a diagnostic adjunct to the routine cytologic evaluation of malignancy in peritoneal washings; reactivity with MAb B72.3 may indicate the need for further evaluation to define the presence of a malignancy or an advanced cancer.