An improved monocyte activation test using cryopreserved pooled human mononuclear cells

Solati, Shabnam; Aarden, Lucien A.; Wouters, Diana

doi:10.1177/1753425915583365

Cited by 19 publications

(8 citation statements)

References 14 publications

(18 reference statements)

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“…The fitness-for-purpose of PBMC cryopreservation has been demonstrated in the scope of preservation of lymphocyte immunophenotypes and proliferative responses [48,49], pyrogenic responses [50,51], establishment of LCLs [52], Th1/Th2 cytokine ratio after stimulation [53], Tcell culture and subsequent polyfunctional antigen-specific CD4 and CD8 T cell responses [16,54], host cell reactivation (HCR) assay [55], and mutagen sensitivity assay for assessment of DNA repair capacity [56]. Cryopreservation does not induce any significant difference in the proportions of T cells expressing CD3 and monocytes/ macrophages expressing CD14 [57].…”

Section: Cryopreservationmentioning

confidence: 99%

Biospecimen Science of Blood for Peripheral Blood Mononuclear Cell (PBMC) Functional Applications

et al. 2019

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Purpose of Review Peripheral blood mononuclear cells (PBMCs) are used in a wide variety of preclinical assays. Preanalytical variations can have a devastating impact on the results. In this review, we list critical preanalytical factors for PBMC-based assays to develop awareness and orientation to the types of sample preparation and storage that one may consider employing. Recent Findings Critical factors during blood collection are the blood collection tube and anticoagulant, possible stabilizer used, and the pre-isolation blood storage temperature and time. During PBMC isolation, critical factors are the isolation method, density gradient or magnetic sorting, use of barrier, possible RBC lysis, and centrifuge type. During cryopreservation, attention is needed for the cryomedium type and temperature, freezing device and program, cell concentration, and the long-term storage temperature. During the thawing process, the thawing procedure/device used and wash medium temperature are critical. Summary To avoid biased results in PBMC assays, these critical preanalytical factors must be standardized and/or documented. Additionally, participation in external quality assurance programs is strongly recommended.

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Section: Cryopreservationmentioning

confidence: 99%

Biospecimen Science of Blood for Peripheral Blood Mononuclear Cell (PBMC) Functional Applications

et al. 2019

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“…Although the isolation of PBMC from whole blood takes considerably longer than using fresh blood and also bears the possibility of unspecific stimulation of the blood cells, it still offers the normalization of the cell numbers and cryopreservation. This was recently shown by Solati and co‐workers . After isolation of PMBC the cells were either used freshly or cryopreserved from individual donors or pooled.…”

Section: Discussionmentioning

confidence: 96%

Speeding up pyrogenicity testing: Identification of suitable cell components and readout parameters for an accelerated monocyte activation test (MAT)

Stoppelkamp¹,

Würschum²,

Stang³

et al. 2016

Drug Testing and Analysis

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Pyrogen testing represents a crucial safety measure for parental drugs and medical devices, especially in direct contact with blood or liquor. The European Pharmacopoeia regulates these quality control measures for parenterals. Since 2010, the monocyte activation test (MAT) has been an accepted pyrogen test that can be performed with different human monocytic cell sources: whole blood, isolated monocytic cells or monocytic cell lines with IL1β, IL6, or TNFα as readout cytokines. In the present study, we examined the three different cell sources and cytokine readout parameters with the scope of accelerating the assay time. We could show that despite all cell types being able to detect pyrogens, primary cells were more sensitive than the monocytic cell line. Quantitative real-time PCR revealed IL6 mRNA transcripts having the largest change in Ct-values upon LPS-stimulation compared to IL1β and TNFα, but quantification was unreliable. IL6 protein secretion from whole blood or peripheral blood mononuclear cells (PBMC) was also best suited for an accelerated assay with a larger linear range and higher signal-to-noise ratios upon LPS-stimulation. The unique combination with propan-2-ol or a temperature increase could additionally increase the cytokine production for earlier detection in PBMC. The increased incubation temperature could finally increase not only responses to lipopolysaccharides (LPS) but also other pyrogens by up to 13-fold. Therefore, pyrogen detection can be accelerated considerably by using isolated primary blood cells with an increased incubation temperature and IL6 as readout. These results could expedite assay time and thus help to promote further acceptance of the MAT. Copyright © 2016 John Wiley & Sons, Ltd.

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“…[20][21][22] Though cryopreserved PBMC was not involved as a cell-source in these international validations, advantages of MAT based on cryopreserved PBMC have been demonstrated in recent years in research and development. 17…”

Section: Discussionmentioning

confidence: 99%

The monocyte activation test detects potentiated cytokine release resulting from the synergistic effect of endotoxin and non-endotoxin pyrogens

Solati¹,

Zhang²,

Timman³

2022

Innate Immun

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Pyrogens are classified in two groups, endotoxin pyrogens and non-endotoxin pyrogens (NEPs). The presence of either in parenteral pharmaceuticals or medical devices can cause severe harm to subjects, and when occurring in combination, synergistic potentiation effects can occur. As the standard in vitro pyrogen test, the Limulus Amebocyte Lysate (LAL) assay can detect LPS only, an endotoxin, but not NEPs. We tested whether the Monocyte Activation Test (MAT) that measures IL-6 induction, is suited for detecting synergistic pyrogen effects. Here we show that MAT reliably detects the NEPs heat-killed Staphylococcus aureus, R848 and lipoteichoic acid, in addition to LPS. When combinations of these pyrogens were tested, a potentiation of IL-6 production was seen beyond an additive effect, apparently reflecting on in-vivo synergisms. The current study therefore demonstrates that MAT not only is a reliable and reproducible assay for the sensitive detection of both endotoxin and non-endotoxin pyrogens, but also for identifying synergistic effects when parenteral drugs are contaminated with multiple pyrogens.

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An improved monocyte activation test using cryopreserved pooled human mononuclear cells

Cited by 19 publications

References 14 publications

Biospecimen Science of Blood for Peripheral Blood Mononuclear Cell (PBMC) Functional Applications

Biospecimen Science of Blood for Peripheral Blood Mononuclear Cell (PBMC) Functional Applications

Speeding up pyrogenicity testing: Identification of suitable cell components and readout parameters for an accelerated monocyte activation test (MAT)

The monocyte activation test detects potentiated cytokine release resulting from the synergistic effect of endotoxin and non-endotoxin pyrogens

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