Exosomal miR-16-5p as a target for malignant mesothelioma.

Malignant mesothelioma (MM) is an asbestos-induced cancer arising on the mesothelial surface of organ cavities. MM is essentially incurable without a means of early diagnosis and no successful standard of care. These facts indicate a deep chasm of knowledge that needs to be filled. Our group recently delved into MM tumor biology from the perspective of exosome-contained microRNAs (miRNAs). We discovered that the most abundant miRNAs in MM cancer exosomes were tumor suppressors, particularly miR-16-5p. This observation lead us to hypothesize that MM cells preferentially secreted tumor-suppressor miRNAs via exosomes. Through separate avenues of potential therapeutic advance, we embarked on an innovative strategy to kill MM tumor cells. We employed small molecule inhibitors to block exosome secretion, thereby reducing miR-16-5p exosome loss and replenishing cellular miR-16-5p leading to reduced tumorigenic capacity and miR-16-5p target oncoproteins CCND1 and BCL2. Additionally, we force-fed MM tumor exosomes back to MM tumor cells, which led to cell death, and a reduction in the same oncoproteins. We recapitulated these results with direct transfection of miR-16-5p, confirmed that this is a cancer-cell specific effect, and elucidated a part of the miR-16-5p mechanism of exosome loading.

Glucose-6-phosphate dehydrogenase enzyme stability in filter paper dried blood spots.

OBJECTIVE: Prior to initial distribution of Glucose-6-phosphate dehydrogenase (G6PD) proficiency testing (PT) materials, we evaluated G6PD enzyme stability in dried blood spots (DBS) under various temperature and humidity environments to develop storage and usage guidelines for our new materials. DESIGN & METHODS: We prepared fresh G6PD-normal DBS materials and conducted stability evaluations of daily use and short and long-term storage under various temperature and humidity environments. RESULTS: G6PD DBS PT materials retained 92% of initial activity after 30days of use at 4°C. Materials stored at -20°C and 4°C with desiccant for 30days retained 95% and 90% of initial activity, respectively. When stored for one year at -20°C or six months at 4°C specimens retained >90% of initial activity. Specimens stored at 37°C with desiccant lost 10% activity in three days. At the end of 30days, specimens stored under 'Extreme'-humidity >50% without desiccant- conditions at 37°C assayed below the NSQAP cut off for G6PD. Humidity exacerbated loss of enzyme activity with increasing temperature and time duration. CONCLUSION: Data suggest that G6PD PT materials can be stored at 4°C and used for up to one month and can be stored at -20°C for one year and yield >90% enzyme activity. Exposure to warm temperatures, especially with elevated humidity, should be avoided. Desiccant should always be used to mitigate humidity effects.

Influence of Hematocrit and Total-Spot Volume on Performance Characteristics of Dried Blood Spots for Newborn Screening.

Dried blood spots (DBS) have been used in newborn screening (NBS) tests for over 50 years. The Newborn Screening Quality Assurance Program (NSQAP) at the Centers for Disease Control and Prevention (CDC) conducted studies to assess the individual impacts of hematocrit and total-spot volume on characteristics of DBS samples. Per-punch serum volumes decreased 27%, RBC volumes more than doubled, absorption times increased over 300%, and spot diameters decreased marginally between the hematocrits of 40% to 65%. Per-punch serum and RBC volumes decreased logarithmically with lowering total-spot volumes. Patient hematocrit is an uncontrollable variable and inevitably affects the resulting punch from a DBS sample. It may be possible, though, to identify samples that fall outside of an acceptable range by noting certain physical characteristics of the DBS.

Performance properties of filter paper devices for whole blood collection.

BACKGROUND: The Newborn Screening Quality Assurance Program at the Centers for Disease Control and Prevention assesses the adherence to established performance standards of manufactured lots of whole blood filter paper collection devices that are registered by the US FDA. We examined 26 newborn screening analytes measured from blood applied to filter papers from two FDA-cleared sources, Whatman(®) Grade 903 and Ahlstrom Grade 226. The dried blood spots contained analytes at both single levels and dose-response series. RESULTS: We observed overlap at one standard deviation for each analyte, with no more than 4-5% difference between the papers. CONCLUSION: The data demonstrated similarities of analyte recovery between the papers, indicating comparability of the devices for newborn screening and other applications.

Circulating immunoreactive cardiac troponin forms determined by gel filtration chromatography after acute myocardial infarction.

BACKGROUND: Cardiac troponin I (cTnI) and cTnT measurements are used in the diagnosis of acute myocardial infarction (AMI). Together with troponin C (TnC), the cTnI and cTnT forms make up the ternary cTnT-cTnI-TnC (TIC) complex found within myocardium. Whether cTn occurs in the circulation after AMI as ternary TIC, binary cTnI-TnC (IC) complexes, or free troponin forms has not been thoroughly investigated. METHODS: Blood samples from 10 AMI patients were collected at hospital admission and then at 12, 24, and 48 h after onset of chest pain. Serum was subjected to gel filtration chromatography and cTnT (Roche cTnT) and cTnI (Siemens Centaur UltraTnI and Beckman Access AccuTnI) concentrations were measured in the gel filtration chromatography fractions. RESULTS: cTnT was present predominantly as free cTnT and cTnI as binary IC complex. These 2 forms were present at every time point. Lesser quantities of TIC complex (6%-32% of total cTnT and <50% of total cTnI) were detected in 4 patients at varying times. Minor quantities of a high molecular mass form of cTnI were detected occasionally. No free cTnI was found. Both cTnI assays identified a similar pattern of cTnI forms. CONCLUSIONS: After AMI, cTnI is present in serum as TIC and IC complexes. cTnT may be present as a combination of TIC and free cTnT or exclusively as free cTnT.

Preliminary proficiency testing results for succinylacetone in dried blood spots for newborn screening for tyrosinemia type I.

BACKGROUND: Succinylacetone (SUAC) is the primary metabolite accumulated in tyrosinemia type I--an inborn error of metabolism that, if untreated, can cause death from liver failure during the first months of life. Newborn screening laboratories measure SUAC in dried blood spot (DBS) samples to detect asymptomatic tyrosinemia type I. We used panels of SUAC-enriched DBSs to compare and evaluate the performance of these screening tests. METHODS: We prepared sets of DBS materials enriched with predetermined SUAC concentrations and distributed samples of these materials, along with a screening practices questionnaire, to laboratories that perform SUAC tests. We compared their reported SUAC concentrations and questionnaire responses to identify screening practices that affect SUAC test outcomes. RESULTS: Data from 2 pilot surveys showed large differences among laboratories in SUAC recoveries, reproducible within-laboratory recoveries, and stable performance of the DBS materials. Results from 257 proficiency test analyses contained a total of 6 false-negative misclassifications. Reported recoveries of added SUAC ranged from 0 to >200%. Low-biased SUAC recoveries were associated with 1 method used by 5 laboratories. All laboratories that reported SUAC recoveries > or =100% used DBS matrix calibrators. CONCLUSIONS: The wide ranges of SUAC concentrations reported for pilot and proficiency testing specimens demonstrate a need to harmonize quantitative results among laboratories. Although DBS matrix calibrators are important for optimizing SUAC recoveries, the preparation of these calibrators is not standardized among laboratories. Certified DBS-based SUAC calibrators are needed for accuracy and harmonization.

Cardiac troponin T circulates in the free, intact form in patients with kidney failure.

BACKGROUND: The clinical significance of the increased concentrations of cardiac troponins observed in patients with end stage renal disease (ESRD) in the absence of an acute coronary syndrome (ACS) is controversial. One proposed explanation is that immunoreactive fragments of cardiac troponin T (cTnT) accumulate in ESRD. We used gel-filtration chromatography (GFC) to ascertain whether fragments of cTnT, which could cross-react in the commercial diagnostic immunoassay (Roche Diagnostics), were the cause of the increased cTnT in the serum of patients with ESRD. METHODS: We subjected sera from ESRD patients (n = 21) receiving dialysis and having increased cTnT concentrations to size-separation GFC. We detected cTnT in the chromatography fractions by use of the same antibodies used in the commercial assay for serum cTnT. RESULTS: In all patients, cTnT immunoreactivity eluted as a major, homogeneous peak in an identical position between the peaks of serum prolactin [relative molecular mass (Mr) 23,000] and albumin (Mr 67,000): the elution pattern of cTnT in samples obtained from ACS patients was identical to that of the ESRD patients. There was no evidence that low-molecular-mass (Mr < 23,000) cTnT fragments were the cause of the increased cTnT in the patients studied. CONCLUSIONS: The form of cTnT observed in the serum of patients with kidney failure and immunoreactive in the diagnostic assay is predominantly the free intact form, as in patients with ACS. Our data are consistent with the view that circulating cTnT in renal failure reflects cardiac pathology.

More than meets the eye: how examiner training affects the reliability of the MacNeill-Lichtenberg decision tree in geriatric rehabilitation patients.

OBJECTIVE: To examine the effect of examiner training on the test-retest reliability of the MacNeill-Lichtenberg Decision Tree (MLDT), a tool for guiding clinicians' decision making for referrals for mental health problems. DESIGN: Correlational analyses and chi-squares were used to examine the influence of demographic variables on MLDT performance and the test-retest reliability of its cognitive and affective components. SETTING: Rehabilitation unit of a large, freestanding, urban hospital. PARTICIPANTS: In study 1, 39 older, medical rehabilitation patients consecutively referred to the neuropsychology service. In study 2, 57 older, consecutively admitted medical rehabilitation patients. INTERVENTIONS: In study 1, patients underwent testing with the MLDT by a novice examiner. In study 2, patients were tested by trained examiners. Both sets of results were compared with those obtained by experienced examiners. MAIN OUTCOME MEASURES: The temporal stability of participants' performance on the cognitive and affective components of the MLDT was compared between the 2 studies. RESULTS: Training was associated with high test-retest reliability on both the cognitive and affective components. A lack of training was associated with reduced reliability in depression screening. CONCLUSION: These findings support the use of the MLDT as a mental health triage tool for older adults in inpatient medical settings. Its use is dependent on training and accurate administration.