Dataset Information

Comparison of Flow Cytometric Methods for the Enumeration of Residual Leucocytes in Leucoreduced Blood Products: A Multicenter Study.

ABSTRACT: The BD FACSVia™ System features novel designs in hardware, software, and instrument QC. We compared the performance of the BD FACSVia System using the BD Leucocount™ kit with the BD FACSCalibur™ flow cytometer. Leucoreduced platelet (PLT, n = 252) and red blood cell (RBC, n = 278) specimens were enrolled at four sites. Each specimen was stained in four tubes using the BD Leucocount kit reagents and acquired on the two systems. BD Leucocount Control cells (high and low) were used to evaluate the inter-site reproducibility on the BD FACSVia System at three sites over 20 days. Deming regression and Bland-Altman analysis were performed to determine the WBC absolute counts on the BD FACSVia System vs. the BD FACSCalibur system. Assay accuracy for the range of 0-350 WBCs/µl was adequate. For samples with <25 WBCs/µl, the bias with 95% limits of agreement was 0.136 (-1.897 to 2.169) WBC/µl for PLTs (n = 184) and 0.170 (-2.025 to 2.365) WBC/µl for RBCs (n = 193). For inter-site reproducibility, the CV% was 6.46% (upper 95% CI 7.16%) for the PLT high control and 9.49% (10.52%) for the PLT low control. The CV% was 7.51% (8.32%) for the RBC high control and 10.76% (11.92%) for the RBC low control. The BD FACSVia System reported equivalent results of WBC absolute counts for leucoreduced PLT and RBC samples compared to the BD FACSCalibur system. The inter-laboratory reproducibility of the BD FACSVia System met study specifications. © 2018 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.

SUBMITTER: Zeng Y

PROVIDER: S-EPMC5947640 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Comparison of Flow Cytometric Methods for the Enumeration of Residual Leucocytes in Leucoreduced Blood Products: A Multicenter Study.

Zeng Yang Y Dabay Michelle M George Virginia V Seetharaman Shalini S de Arruda Indig Monika M Graminske Sharon S Kimpel Nicole N Schmidt Anna A Boerner Amanda A Paradiso Sarai S Delman Tatyana T Li Yunyao Y Litvak Viktoriya V Oreizy Farzad F Chen Angela A Saleminik Maryam M Mosqueda Fred F Lin Anna A Judge Kevin K

Cytometry. Part A : the journal of the International Society for Analytical Cytology 20180118 4

The BD FACSVia™ System features novel designs in hardware, software, and instrument QC. We compared the performance of the BD FACSVia System using the BD Leucocount™ kit with the BD FACSCalibur™ flow cytometer. Leucoreduced platelet (PLT, n = 252) and red blood cell (RBC, n = 278) specimens were enrolled at four sites. Each specimen was stained in four tubes using the BD Leucocount kit reagents and acquired on the two systems. BD Leucocount Control cells (high and low) were used to evaluate the ...[more]

PMID: 29345745

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Project description:Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the β-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists' discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Project description:Essentials The clinical enumeration of microparticles (MPs) is hampered by a lack of standardization. A new strategy to standardize MP counts by flow cytometry was evaluated in a multicenter study. No difference was found between instruments using forward or side scatter as the trigger parameter. This study demonstrated that beads can be used as a standardization tool for MPs. Click to hear the ISTH Academy's webinar on microvesicles SUMMARY: Background Microparticles (MPs) are extracellular vesicles resulting from the budding of cellular membranes that have a high potential as emergent biomarkers; however, their clinical relevance is hampered by methodological enumeration concerns and a lack of standardization. Flow cytometry (FCM) remains the most commonly used technique with the best capability to determine the cellular origin of single MPs. However, instruments behave variably depending on which scatter parameter (forward (FSC) or side scatter (SSC)) provides the best resolution to discriminate submicron particles. To overcome this problem, a new approach, based on two sets of selected beads adapted to FSC or SSC-optimized instruments, was recently proposed to reproducibly enumerate platelet-derived MP counts among instruments with different optical systems. Objective The objective was to evaluate this strategy in an international workshop that included 44 laboratories accounting for 52 cytometers of 14 types. Methods/Results Using resolution capability and background noise level as criteria to qualify the instruments, the standardization strategy proved to be compatible with 85% (44/52) of instruments. All instruments correctly ranked the platelet MP (PMP) levels of two platelet-free plasma samples. The inter-laboratory variability of PMP counts was 37% and 28% for each sample. No difference was found between instruments using forward or side-scattered light as the relative sizing parameter. Conclusions Despite remaining limitations, this study is the first to demonstrate a real potential of bead-based strategies for standardization of MP enumeration across different FCM platforms. Additional standardization efforts are still mandatory to evaluate MPs' clinical relevance at a multicenter level.

Dataset Information

Comparison of Flow Cytometric Methods for the Enumeration of Residual Leucocytes in Leucoreduced Blood Products: A Multicenter Study.

Publications

Comparison of Flow Cytometric Methods for the Enumeration of Residual Leucocytes in Leucoreduced Blood Products: A Multicenter Study.

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