Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat.

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR). The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS). The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.

Optimized processing for pathogen inactivation of double-dose buffy-coat platelet concentrates: maintained in vitro quality over 7-day storage.

BACKGROUND AND OBJECTIVE: Efficient pathogen inactivation (PI) offers the possibility of increasing the number of buffy coats per pool without the concurrent increased risk of pathogen transmission. Here, we describe the findings of in vitro analyses of platelets from pools of eight buffy coats treated with amotosalen and UVA light (INTERCEPT Blood System for Platelets) using INTERCEPT disposable processing sets with plastic materials sourced from alternate suppliers and split afterwards to obtain two therapeutic transfusion doses. METHODS: Double-dose platelet concentrates were prepared from pools of eight buffy coats in additive solution (SSP+) using either previous 6-lead or new 8-lead pooling sets and PI processing sets in previous or alternate supplier sourced plastics (AS). Platelets were treated with the INTERCEPT Blood System then stored for up to 7 days and tested for in vitro quality. RESULTS: All tested units (n = 30) were in conformity with European guidelines. Using AS sets more effectively maintained glucose reserves (P < 0·01), reduced lactate production (P < 0·01), reduced CD62P expression (P < 0·01) and downregulated levels of surface CD42b (P < 0·01) overtime. AS set maintained JC-positive cells (NS) between day 2 and day 7 and sustained platelet integrin activation (PAC-1) between day 2 and day 7 (NS). Overall sCD40L and PGDF accumulated in an equivalent way (P < 0·01) within series. SUMMARY/CONCLUSIONS: In summary, our data demonstrate that PI treatment using AS sets, in combination with the new pooling set for double-dose platelet preparation, maintained the platelet in vitro quality over 7 days of storage.

Apheresis buffy coat collection without photoactivation has no effect on apoptosis, cell proliferation, and total viability of mononuclear cells collected using photopheresis systems.

BACKGROUND: Extracorporeal photopheresis (ECP) has been approved for the treatment of advanced cutaneous T-cell lymphoma since 1988. While the precise mechanisms resulting in clinical effects are not fully understood, the photoactivation of mononuclear cells (MNCs) using ultraviolet A (UVA) light and methoxsalen is believed to be the predominant initiating process. The effects of MNC passage through the instrument without photoactivation are unknown. The objective of this study was to evaluate the effect of cell processing through the photopheresis instruments on MNCs. STUDY DESIGN AND METHODS: Fourteen healthy male subjects underwent one simulated ECP procedure without reinfusion of buffy coats (BCs) in a two-center, open-label, prospective trial. Baseline peripheral blood BC, apheresis-separated untreated BC (BC1), and photoactivated BC (BC2) were evaluated in culture for viability by dye exclusion, apoptosis by annexin V binding, and cell proliferation response to phytohemagglutinin (PHA) stimulation by bromodeoxyuridine (BrdU) incorporation. RESULTS: Photoactivation (BC2) resulted in 88% expression of annexin V by Day 1 of culture compared with 37 and 39% for baseline and untreated BC1. Cell viability by propidium iodide exclusion was reduced to 10% in BC2 on Day 1 versus 65 and 60% for baseline and BC1. The proliferative response to PHA stimulation was 97% inhibited in the photoactivated BC2. CONCLUSIONS: These results demonstrate that the mechanical processes used for cell separation and processing of the BC in the absence of photoactivation do not induce a significant amount of apoptosis compared to the standard ECP with methoxsalen and UVA photoactivation.

In vitro comparison between gamma-irradiated cryopreserved and Day 7 liquid-stored buffy coat-derived platelet components.

BACKGROUND: Cryopreserved platelet (PLT) components stored at -80°C in 5% to 6% dimethyl sulfoxide (DMSO) demonstrate enhanced hemostatic activity. Alterations in PLT surface glycoprotein expression and release of procoagulant microparticles during the freeze/thaw cycle result in PLT activation. Nothing is known of the effect of gamma irradiation on the in vitro quality of reconstituted cryopreserved PLTs. STUDY DESIGN AND METHODS: Gamma-irradiated (25-50 Gy) buffy coat-derived PLT components were either stored at room temperature for 7 days (the current expiry in New Zealand) or cryopreserved at -80°C using 5% to 6% DMSO. Cryopreserved PLTs were thawed at 37°C and reconstituted in ABO-identical plasma or PAS-E and compared to Day 7 gamma-irradiated liquid-stored PLTs. In vitro assays were performed to assess glycoprotein expression, PLT functionality and soluble cytokine release. RESULTS: Cryopreserved PLTs after thawing and reconstitution in ABO-matched plasma or PAS-E displayed differing recoveries (82.7 and 75.9%, respectively). Key expression levels of glycoproteins GPIbα (CD42b) and GPIIb (CD41a) were reduced. Cryopreserved PLTs retained the ability to form an effective functional clot, while showing accelerated initiation of clot formation (R-time) compared to Day 7 gamma-irradiated liquid-stored PLTs. CONCLUSION: Gamma-irradiated buffy coat-derived liquid-stored and cryopreserved PLTs have distinctly differing phenotypes. Cryopreserved PLTs reconstituted in ABO plasma have enhanced clot strength driven by coagulation factors and fibrinogen levels not present in PAS-E. Irradiated cryopreserved PLTs maintain a similar in vitro quality profile and hemostatic behavior to previously published, nonirradiated cryopreserved PLTs.