Dysfunction of the muscles of ambulation contributes to exercise intolerance in chronic obstructive pulmonary disease (COPD). Men with COPD have high prevalence of low testosterone levels, which may contribute to muscle weakness. We determined effects of testosterone supplementation (100 mg of testosterone enanthate injected weekly) with or without resistance training (45 minutes three times weekly) on body composition and muscle function in 47 men with COPD (mean FEV(1) = 40% predicted) and low testosterone levels (mean = 320 ng/dl). Subjects were randomized to 10 weeks of placebo injections + no training, testosterone injections + no training, placebo injections + resistance training, or testosterone injections + resistance training. Testosterone injections yielded a mean increase of 271 ng/dl in the nadir serum testosterone concentration (to the middle of the normal range for young men). The lean body mass (by dual-energy X-ray absorptiometry) increase averaged 2.3 kg with testosterone alone and 3.3 kg with combined testosterone and resistance training (p < 0.001). Increase in one-repetition maximum leg press strength averaged 17.2% with testosterone alone, 17.4% with resistance training alone, and 26.8% with testosterone + resistance training (p < 0.001). Interventions were well tolerated with no abnormalities in safety measures. Further studies are required to determine long-term benefits of adding testosterone supplementation and resistance training to rehabilitative programs for carefully screened men with COPD and low testosterone levels.

BackgroundCryopreservation of peripheral blood mononuclear cells (PBMCs) is a common and essential practice in conducting research. There are different reports in the literature as to whether cryopreserved PBMCs need to only be stored ≤ −150 °C or can be stored for a specified time at −80 °C. Therefore, we performed gene expression analysis on cryopreserved PBMCs stored at both temperatures for 14 months and PBMCs that underwent temperature cycling 104 times between these 2 storage temperatures. Real-time RT-PCR was performed to confirm the involvement of specific genes associated with identified cellular pathways. All cryopreserved/stored samples were compared to freshly isolated PBMCs and between storage conditions.ResultsWe identified a total of 1,367 genes whose expression after 14 months of storage was affected >3 fold in PBMCs following isolation, cryopreservation and thawing as compared to freshly isolated PBMC aliquots that did not undergo cryopreservation. Sixty-six of these genes were shared among two or more major stress-related cellular pathways (stress responses, immune activation and cell death). Thirteen genes involved in these pathways were tested by real-time RT-PCR and the results agreed with the corresponding microarray data. There was no significant change on the gene expression if the PBMCs experienced brief but repetitive temperature cycling as compared to those that were constantly kept ≤ −150 °C. However, there were 18 genes identified to be different when PBMCs were stored at −80 °C but did not change when stored < −150 °C. A correlation was also found between the expressions of 2′–5′- oligoadenylate synthetase (OAS2), a known interferon stimulated gene (IFSG), and poor PBMC recovery post-thaw. PBMC recovery and viability were better when the cells were stored ≤ −150 °C as compared to −80 °C.ConclusionsNot only is the viability and recovery of PBMCs affected during cryopreservation but also their gene expression pattern, as compared to freshly isolated PBMCs. Different storage temperature of PBMCs can activate or suppress different genes, but the cycling between −80 °C and −150 °C did not produce significant alterations in gene expression when compared to PBMCs stored ≤ −150 °C. Further analysis by gene expression of various PBMC processing and cryopreservation procedures is currently underway, as is identifying possible molecular mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-016-0144-1) contains supplementary material, which is available to authorized users.

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The ranked listing of 170 preanalytic variables produced can be used as a guide for site-specific implementation into patient care and/or research biorepository processes. Conclusions.-In our collective experience, it is often difficult to choose which of the many preanalytic variables to attach to any specific set of biospecimens used for patient care and/or research. The provided ranked list should aid in the selection of preanalytic variables for a given biospecimen collection.

The use of cryopreserved peripheral blood mononuclear cells (PBMCs) has proven critical in both clinical and biomedical applications. While utilized, in numerous settings, the functionality of frozen PBMCs is often limited, and a disconnect exists between measured viability and outcome. In this study we investigated parameters affecting outcome including storage protocol, freeze media, and assessment assay. PBMCs were isolated from seven healthy donors and cryopreserved in: (1) a media-based cocktail (SAIC), (2) CryoStor 7.5 (CS7.5), and (3) CryoStor 7.5 plus caspase inhibitors (CS7.5 ؉ Inh). All samples were stored in vapor phase liquid nitrogen (static) with replicates exposed to a thermal cycling regime, which mimicked typical sample handling (cycle). Viability was assessed immediately postthaw by trypan blue. Cryopreservation-induced delayed-onset cell death (CIDOCD) was evaluated postthaw using the Vybrant ® Apoptosis Assay (VAA) that differentiated viability, apoptosis, and necrosis by fluorescent microscopy. Analysis of static stored PBMCs in CS7.5 demonstrated improved viability 1 h postthaw (82%-90%) compared to SAIC samples (60%-72%). Both cryococktails performed similarly after 24 h postthaw, yet fewer surviving PBMCs were evident, especially when assessed by VAA (SAIC: 31%; CS7.5: 36%). Replicate PBMC aliquots subjected to temperature cycling did not appear to have altered viability at 1 and 24 h postthaw (trypan blue). However, VAA showed that the SAIC cryococktail yielded improved viability at 1 h postthaw (61% vs. 51.8%, respectively) whereas by 24 h both the SAIC and CS7.5 samples were similar (ϳ20%). CIDOCD assessment revealed significant levels of both necrosis and apoptosis at 4 h and 8 h across all conditions. The observed decline in viability in the cycled samples was a result of increased apoptosis. Finally, the incorporation of caspase inhibitors did not improve static sample viability, but did significantly reduce the negative effect of temperature cycling. These preliminary investigations demonstrate that CIDOCD plays a significant role in PBMC cryopreservation failure, and that temperature fluctuations influence the overall level. Further, these studies demonstrate the importance of using multiple viability assessment techniques in evaluating PBMC cryococktail performance. 189

Studies have examined the associations between cancers and circulating 25-hydroxyvitamin D [25(OH)D], but little is known about the impact of different laboratory practices on 25(OH)D concentrations. We examined the potential impact of delayed blood centrifuging, choice of collection tube, and type of assay on 25(OH)D concentrations. Blood samples from 20 healthy volunteers underwent alternative laboratory procedures: four centrifuging times (2, 24, 72, and 96 h after blood draw); three types of collection tubes (red top serum tube, two different plasma anticoagulant tubes containing heparin or EDTA); and two types of assays (DiaSorin radioimmunoassay [RIA] and chemiluminescence immunoassay [CLIA/LIAISON®]). Log-transformed 25(OH)D concentrations were analyzed using the generalized estimating equations (GEE) linear regression models. We found no difference in 25(OH)D concentrations by centrifuging times or type of assay. There was some indication of a difference in 25(OH)D concentrations by tube type in CLIA/LIAISON®-assayed samples, with concentrations in heparinized plasma (geometric mean, 16.1 ng ml−1) higher than those in serum (geometric mean, 15.3 ng ml−1) (p = 0.01), but the difference was significant only after substantial centrifuging delays (96 h). Our study suggests no necessity for requiring immediate processing of blood samples after collection or for the choice of a tube type or assay.

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Studies have examined the associations between cancers and circulating 25-hydroxyvitamin D [25 (OH)D], but little is known about the impact of different laboratory practices on 25(OH)D