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Ki-67 staining for determination of rhesus macaque T cell proliferative responses ex vivo.

ABSTRACT: The capacity for robust proliferation upon re-infection is a hallmark of adaptive immunity and the basis of vaccination. A widely used animal model for the study of human disease is the rhesus macaque (RM), where capacity for proliferation can be assessed ex vivo using carboxyfluorescein succinimidyl ester (CFSE)-based dilution assays. However, we show over the course of the standard ex vivo proliferation assay that CFSE-labeling at commonly used dye concentrations induces significant cell death, but that this phenomenon is dose-dependent. Here, we describe an alternative semiquantitative method for estimating T cell proliferative responses that avoids the putative biases associated with chemical modification. RM peripheral blood mononuclear cells were stimulated ex vivo with cognate peptides for 5 days, immunostained for intracellular Ki-67, and then analyzed by flow cytometry. We describe a gating strategy using Ki-67 and side light scatter, also a marker of blastogenesis, which correlates strongly with data from CFSE dilution. We show that this method is a valid tool for measuring RM antigen-specific cellular proliferation ex vivo and can be used as an alternative to CFSE dilution assays.

SUBMITTER: Shedlock DJ

PROVIDER: S-EPMC2939446 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Ki-67 staining for determination of rhesus macaque T cell proliferative responses ex vivo.

Shedlock Devon J DJ Talbott Kendra T KT Morrow Matthew P MP Ferraro Bernadette B Hokey David A DA Muthumani Karuppiah K Weiner David B DB

Cytometry. Part A : the journal of the International Society for Analytical Cytology 20100301 3

The capacity for robust proliferation upon re-infection is a hallmark of adaptive immunity and the basis of vaccination. A widely used animal model for the study of human disease is the rhesus macaque (RM), where capacity for proliferation can be assessed ex vivo using carboxyfluorescein succinimidyl ester (CFSE)-based dilution assays. However, we show over the course of the standard ex vivo proliferation assay that CFSE-labeling at commonly used dye concentrations induces significant cell death ...[more]

PMID: 20104580

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Project description:BACKGROUND:The scoring of Ki-67 is highly relevant for the diagnosis, classification, prognosis, and treatment in breast invasive ductal carcinoma (IDC). Traditional scoring method of Ki-67 staining followed by manual counting, is time-consumption and inter-/intra observer variability, which may limit its clinical value. Although more and more algorithms and individual platforms have been developed for the assessment of Ki-67 stained images to improve its accuracy level, most of them lack of accurate registration of immunohistochemical (IHC) images and their matched hematoxylin-eosin (HE) images, or did not accurately labelled each positive and negative cell with Ki-67 staining based on whole tissue sections (WTS). In view of this, we introduce an accurate image registration method and an automatic identification and counting software of Ki-67 based on WTS by deep learning. METHODS:We marked 1017 breast IDC whole slide imaging (WSI), established a research workflow based on the (i) identification of IDC area, (ii) registration of HE and IHC slides from the same anatomical region, and (iii) counting of positive Ki-67 staining. RESULTS:The accuracy, sensitivity, and specificity levels of identifying breast IDC regions were 89.44, 85.05, and 95.23%, respectively, and the contiguous HE and Ki-67 stained slides perfectly registered. We counted and labelled each cell of 10 Ki-67 slides as standard for testing on WTS, the accuracy by automatic calculation of Ki-67 positive rate in attained IDC was 90.2%. In the human-machine competition of Ki-67 scoring, the average time of 1 slide was 2.3?min with 1 GPU by using this software, and the accuracy was 99.4%, which was over 90% of the results provided by participating doctors. CONCLUSIONS:Our study demonstrates the enormous potential of automated quantitative analysis of Ki-67 staining and HE images recognition and registration based on WTS, and the automated scoring of Ki67 can thus successfully address issues of consistency, reproducibility and accuracy. We will provide those labelled images as an open-free platform for researchers to assess the performance of computer algorithms for automated Ki-67 scoring on IHC stained slides.

Project description:Background p16/Ki-67 dual immunocytochemical staining (DS) is considered easy to interpret if evaluators are properly trained, however, there is no consensus on what constitutes proper training. In the present study we evaluated a protocol for teaching DS evaluation on students inexperienced in cervical cytology. Methods Initial training on 40 DS conventional smears was provided by a senior cytotechnologist experienced in such evaluation. Afterwards, two students evaluated 118 cases. Additional training consisted mainly of discussing discrepant cases from the first evaluation and was followed by evaluation of new 383 cases. Agreement and accuracy of students' results were compared among the participants and to the results of the reference after both evaluations. We also noted time needed for evaluation of one slide as well as intra-observer variability of the teacher's results. Results At the end of the study, agreement between students and reference was higher compared to those after initial training (overall percent agreement [OPA] 81.4% for each student, kappa 0.512 and 0.527 vs. OPA 78.3% and 87.2%, kappa 0.556 and 0.713, respectively). However, accuracy results differed between the two students. After initial training sensitivity was 4.3% points and 2.9% points higher, respectively compared to the reference, while specificity was 30.6% points and 24.4% points lower, respectively, compared to the reference. At the end of the study, the sensitivity reached by one student was the same as that of the reference, while it was 2.6% points lower for the other student. There was a statistically significant difference in specificity between one student and the reference and also between students (16.7 and 15.1% points). Towards the end of the study, one student needed 5.2 min for evaluating one slide while the other needed 8.2 min. The intra-observer variability of the senior cytotechnologist was in the range of "very good" in both arms of the study. Conclusions In teaching DS evaluation, the students' progress has to be monitored using several criteria like agreement, accuracy and time needed for evaluating one slide. The monitoring process has to continue for a while after students reach satisfactory results in order to assure a continuous good performance. Monitoring of teacher's performance is also advisable.

Dataset Information

Ki-67 staining for determination of rhesus macaque T cell proliferative responses ex vivo.

Publications

Ki-67 staining for determination of rhesus macaque T cell proliferative responses ex vivo.

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