Small Blood Samples Research Articles

Microfluidic Point-of-Care Ecarin-Based Clotting and Chromogenic Assays for Monitoring Direct Thrombin Inhibitors

Direct thrombin inhibitors (DTIs), such as bivalirudin and dabigatran, have maintained steady inpatient and outpatient use as substitutes for heparin and warfarin, respectively, because of their high bioavailability and relatively safe “on-therapy” range. Current clinical methods lack the capacity to directly quantify plasma DTI concentrations across wide ranges. At present, the gold standard is the ecarin clotting time (ECT), where ecarin maximizes thrombin activity and clotting time is evaluated to assess DTIs’ anticoagulation capability. This work focused on the development of a microfluidic paper analytic device (µPAD) that can quantify the extent of anticoagulation as well as DTI concentration within a patient’s whole blood sample. Capillary action propels a small blood sample to flow through the nitrocellulose paper channels. Digital images of whole blood migration are then captured by our self-coded Raspberry Pi and/or the Samsung Galaxy S8 smartphone camera. Both the flow length and the blue absorbance from the plasma front on the μPAD were measured, allowing simultaneous, dual assays: ecarin clotting test (ECT) and ecarin chromogenic assay (ECA). Statistically significant (p < .05) changes in flow and absorbance were observed within our translational research study. Currently, there are no quantitative, commercially available point-of-care tests for the ECT and ECA within the United States. Both the ECT and ECA assays could be instrumental to differentiate between supratherapeutic and subtherapeutic incidents during bridging anticoagulant therapy and limit the unwarranted use of reversal agents.

Flow Cytometry Assay to Monitor Effectiveness of Gene Therapy Correction in Peripheral Blood Mononuclear Cells of Gaucher Disease Type I Patients

The most common lysosomal storage disorder is Gaucher disease type I (GD-I) which is caused by a genetic defect in the gene encoding the enzyme beta-glucocerebrosidase (GCase). In GD-I, deficient GCase activity results in accumulation of glucocerebrosidases within macrophages of the reticuloendothelial system.We hypothesize that putative precursors for macrophages, monocytes, in peripheral blood of GD-I patients, represent an excellent target population to monitor the effectiveness of novel curative treatments such as lentiviral vector driven gene correction targeting hematopoietic stem and progenitor cells in clinical setting.Here, we present an improved flow cytometry assay that takes advantage of selective uptake of pentafluorobenzoylamino fluorescein di-β-D-glucopyranoside by the CD14+ monocytes resulting in fluorescence when it is hydrolyzed by active GCase, in the presence of formaldehyde (FA), commonly used for intracellular staining and to inactivate pathogens.Using this substrate uptake/FA fixation method our results show a mean of 33.7% ± 3.6% (X±SD) GCase+ cells in GD-1 patients (n=2) compared to 83.7% ± 9.6% (X±SD) in normal controls (n=5). The mean fluorescence intensity (MFI) of the enzymatic activity showed no overlap between untreated GD-1 patients (3480 ± 49) and controls (7187 ± 595.7). Method specificity was demonstrated by complete inhibition of enzyme activity and fluorescence using 400 mM conduritol-B-epoxide, a GCase specific inhibitor. Next, mixtures of normal donor peripheral blood mononuclear cells (PBMC) and GD-1 PBMC were created in vitro to assess the lower limit of detection. As low as 1% normal donor cells could be reliably distinguished within these mixtures. GCase+ monocytes in the mixtures could be identified and quantified based on a higher MFI compared to GD-1 patient cells.Compared to the conventional 4-MU biochemical method, this approach requires only a small blood sample and minimal processing, is rapid and straightforward, and more importantly allows quantification of GCase enzyme activity at individual cells instead of in a bulk cell lysate. These findings suggest this novel flow cytometry method is sensitive and specific to quantify small increases in GCase activity at single cell level, and thus has potential to monitor in vivo the effectiveness of gene therapy for Gaucher disease Type 1 in patients in real time. Additional sample analysis from GD-1 patients and normal donors are in progress to confirm consistency of the method. DisclosuresNo relevant conflicts of interest to declare.

A lifetime of pulmonary gas exchange.

Pulmonary gas exchange is the primary function of the lung, and during my lifetime, its measurement has passed through many stages. When I was born, many physiologists still believed that the lung secreted oxygen. When I was a medical student, the only way we had to recognize defective gas exchange was whether the patient was cyanosed. The advent of the oximeter soon showed that this sign could be very misleading. A breakthrough was the introduction of blood gas electrodes that could measure the PO 2, PCO 2, and pH of a small sample of arterial blood. It was soon recognized that the commonest cause of hypoxemia was ventilation‐perfusion inequality, and that this could also be responsible for CO 2 retention. In the early days, the understanding of the mechanisms of pulmonary gas exchange relied on graphical analysis because the oxygen and carbon dioxide dissociation curves are nonlinear and interdependent which precluded algebraic methods. However, with the introduction of digital computing, problems that had hitherto been impossible to tackle became amenable to study. A key advance was the development of the Multiple Inert Gas Elimination Technique. Now, noninvasive methods for measuring gas exchange show promise, and the whole subject continues to develop.

Frozen blood clots can be used for the diagnosis of distinct Plasmodium species in man and non-human primates from the Brazilian Atlantic Forest

BackgroundZoonotic infections with epidemic potential, as non-human primate malaria and yellow fever (YF), can overlap geographically. Optimizing a small blood sample for diagnosis and surveillance is of great importance. Blood are routinely collected for YF diagnosis and blood clots usually discarded after serum obtention. Aiming to take sample advantage, the sensitivity of a PCR using extracted DNA from long-term frozen clots from human and non-human primates for detection of Plasmodium spp. in low parasitaemia conditions was assayed.ResultsMalaria diagnosis with DNA extracted from blood clots generated results in agreement with samples obtained with whole blood, including mixed Plasmodium vivax/simium and Plasmodium malariae/brasilianum infections.ConclusionBlood clots from human and non-human primates may be an important and low cost source of DNA for malaria surveillance in the Atlantic Forest.

Dried Blood Spheroids for Dry-State Room Temperature Stabilization of Microliter Blood Samples.

It is well-known that 2D dried blood spots on paper offer a facile sample collection, storage, and transportation of blood. However, large volume requirements, possible analyte instability, and difficult sample recovery plague this method, lowering confidence in analyte quantification. For the first time, we demonstrate a new approach using 3D dried blood spheroids for stabilization of small volume blood samples, mitigating these effects without cold storage. Blood spheroids form on hydrophobic paper, preventing interaction between the sample and paper substrate, eliminating all chromatographic effects. Stability of the enzyme alanine transaminase and labile organic compounds such as cocaine and diazepam were also shown to increase in the spheroid by providing a critical radius of insulation. On-surface analysis of the dried blood spheroids using paper spray mass spectrometry resulted in sub-ng/mL limits of detection for all illicit drugs tested, representing 1 order of magnitude improvement compared with analysis from 2D dried blood spots.

Are we surgeons finding it all too much? Dealing with the pressures of our profession

We all agree that a review of the literature should be systematic and not selective. There is one field in which - I must admit - I have always allowed myself a selective read and that is on the positive effect of red wine on health. I love those steps down to my wine cellar to choose my daily ‘medication’. But now there is no way to ignore it. A proper analysis of 599.912 current drinkers, published in the April issue of the Lancet, has demonstrated that there is no protective effect of alcohol on cardiovascular disease.1 In fact, modern DNA techniques demonstrate that alcohol contributes to poorer health from the first pint! The outcome of the Lancet study provides a basis for further reduction of the health advice for alcohol to a maximum of 100 grams per week for men. Current recommendations are much higher in many countries; for example, in the US, the directive sets a ceiling of 196 grams per week for men. How does this affect our profession? It has been shown that there are more heavy drinkers among physicians than among the general population.2 This reminded me of a situation early in my career -back in the eightees- when I was invited for a live surgical demo. There I was, far away from home. The operating room and instrumentation seemed antique, and the nurses were chatting an unknown language. Twenty orthopods were standing around the OR waiting for me to demonstrate an ankle arthroscopy. In talking with the anesthesiologist, I noticed a heavy smell of alcohol. It was 2PM. What was I to do? I took my host aside, but he reassured me that he ‘works better after a few beers, it’s normal’. I accepted and performed my demo. My public was happy …

Changes to polychlorinated biphenyl (PCB) signatures and enantiomer fractions across different tissue types in Guillemots

Two Guillemot carcases were dissected, each providing 12 discrete tissue samples and 3 samples of partially digested food. One hundred and five PCBs from the 209 PCBs determined by GCxGC-ToFMS were detected. The relative proportions of individual PCBs did not vary greatly within tissue types, although the PCB profile from undigested food could be distinguished. Enantiomer fractions (EFs) were determined for CB-95, CB-136 and CB-149 by GC-HRqToFMS. EFs in the partially digested food were near racemic, with high levels of enrichment for E1 CB-95 in the kidneys and liver (EF of 0.80 and 0.84 respectively). This provides some of the clearest evidence to date that fractionation takes place in the organs where metabolic biotransformation and elimination of PCBs occurs. Our findings also confirm the ability of non-lethal sampling techniques, such as collection of small (<1 g) blood samples, to provide PCB signatures that are representative of an individual organism.

Comparison of Exogenous Ketone Supplements on Latency to CNS Oxygen Toxicity Seizures in Middle‐Aged Rats

Central nervous system oxygen toxicity (CNSOT) seizures manifest as tonic‐clonic seizures and are a limitation of hyperbaric oxygen therapy (HBOT) and Navy SEAL divers breathing 100% oxygen using closed circuit rebreathers. Previous studies showed that ketone ester (1,3‐butanediol acetoacetate diester, KE) delayed latency to seizures in 3 months old Sprague‐Dawley rats. Our present study explores the effect of exogenous ketone supplements on CNSOT seizures across different ketone formulations and dosages in 18‐month‐old Sprague‐Dawley rats that resemble middle age in humans. Rats received one dose of the ketogenic agent by oral gavage 60 minutes before a dive. Treatment groups included control (water), 1,3‐butanediol acetoacetate diester (KE 10g/kg, KE/2 5g/kg), KE+MCT (KE 5g/kg + medium chain triglyceride 5g/kg) and KS+MCT (CaNaBHB 5g/kg + MCT 5g/kg). To simulate a dive, rats were placed on 100% oxygen followed by gas compression to 5 atmospheres absolute (ATA) in a hyperbaric chamber and remained at 5 ATA until exhibiting CNSOT seizures, defined by tonic‐clonic movements. Upon seizure presentation, experiments were immediately terminated and the chamber was decompressed at a rate of 1 ATA/min. A small sample of blood was drawn and tested immediately for glucose and R‐beta‐hydroxybutyrate (βHB) levels. While βHB levels were significantly elevated in all aged‐rat treatment groups, compared to control, the latency to seizures was significantly delayed only in KE (p=0.0003), KE/2 (p=0.023) and KE+MCT (p=0.028) groups. In these groups, the severity of seizures was less than control. The latency to seizures in 18 months old rats were delayed by 179% in KE, 219% in KE+MCT and 55% in KE/2 groups, while only by 29% in KS+MCT group. Previous studies indicate that 3‐month‐old Sprague‐Dawley rats presented 574 ± 116% latency to seizure compared to control indicating age related influence on latency to seizure. Glucose levels were not significantly different from the control group. We can conclude that middle‐aged rats demonstrated increased latency to CNSOT seizures, although at a different rate than younger rats, in response to ketogenic supplements and their combinations, while elevating βHB levels.Support or Funding InformationThis work was supported by ONR Grant N000141310062 (DPD), ONR, Undersea Medicine Program (N000140710890, JBD), ONR, Undersea Medicine Program (N000141612537, JBD).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Human Induced Pluripotent Stem Cell Production and Expansion from Blood using a Non-Integrating Viral Reprogramming Vector.

We describe a method to transform blood lymphocytes into human-induced pluripotent stem cells by delivering four transcription factors with a non-integrative virus. Using human peripheral blood mononuclear cells (PBMCs) as the source cell type for hiPSC reprogramming is advantageous since blood samples are rapidly and safely obtained from nearly-all subjects. Reprogramming factors needed to make hiPSCs are introduced by infecting the PBMCs with non-integrating Sendai virus vectors. Reprogrammed cells can subsequently be quickly expanded for downstream use. In this unit, we present current protocols for the isolation of PBMCs from a small sample of human blood and subsequent viral reprogramming and expansion of PBMCs into hiPSCs. © 2018 by John Wiley & Sons, Inc.

Induction of a central memory and stem cell memory phenotype in functionally active CD4+ and CD8+ CAR T cells produced in an automated good manufacturing practice system for the treatment of CD19+ acute lymphoblastic leukemia.

Relapsed/refractory B-precursor acute lymphoblastic leukemia (pre-B ALL) remains a major therapeutic challenge. Chimeric antigen receptor (CAR) T cells are promising treatment options. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to promote sustained proliferation and persistence after T-cell therapy, constituting essential preconditions for treatment efficacy. Therefore, we set up a protocol for anti-CD19 CAR T-cell generation aiming at high Tcm/Tscm numbers. 100ml peripheral blood from pediatric pre-B ALL patients was processed including CD4+/CD8+-separation, T-cell activation with modified anti-CD3/-CD28 reagents and transduction with a 4-1BB-based second generation CAR lentiviral vector. The process was performed on a closed, automated device requiring additional manual/open steps under clean room conditions. The clinical situation of these critically ill and refractory patients with leukemia leads to inconsistent cellular compositions at start of the procedure including high blast counts and low T-cell numbers with exhausted phenotype. Nevertheless, a robust T-cell product was achieved (mean CD4+ = 50%, CD8+ = 39%, transduction = 27%, Tcm = 50%, Tscm = 46%). Strong proliferative potential (up to > 100-fold), specific cytotoxicity and low expression of co-inhibitory molecules were documented. CAR T cells significantly released TH1 cytokines IFN-γ, TNF-α and IL-2 upon target-recognition. In conclusion, partly automated GMP-generation of CAR T cells from critically small blood samples was feasible with a new stimulation protocol that leads to high functionality and expansion potential, balanced CD4/CD8 ratios and a conversion to a Tcm/Tscm phenotype.

The UAE healthy future study: a pilot for a prospective cohort study of 20,000 United Arab Emirates nationals

BackgroundThe United Arab Emirates (UAE) is faced with a rapidly increasing burden of non-communicable diseases including obesity, diabetes, and cardiovascular disease. The UAE Healthy Future study is a prospective cohort designed to identify associations between risk factors and these diseases amongst Emiratis. The study will enroll 20,000 UAE nationals aged ≥18 years. Environmental and genetic risk factors will be characterized and participants will be followed for future disease events. As this was the first time a prospective cohort study was being planned in the UAE, a pilot study was conducted in 2015 with the primary aim of establishing the feasibility of conducting the study. Other objectives were to evaluate the implementation of the main study protocols, and to build adequate capacity to conduct advanced clinical laboratory analyses.MethodsSeven hundred sixty nine UAE nationals aged ≥18 years were invited to participate voluntarily in the pilot study. Participants signed an informed consent, completed a detailed questionnaire, provided random blood, urine, and mouthwash samples and were assessed for a series of clinical measures. All specimens were transported to the New York University Abu Dhabi laboratories where samples were processed and analyzed for routine chemistry and hematology. Plasma, serum, and a small whole blood sample for DNA extraction were aliquoted and stored at −80 °C for future analyses.ResultsOverall, 517 Emirati men and women agreed to participate (68% response rate). Of the total participants, 495 (95.0%), 430 (82.2%), and 492 (94.4%), completed the questionnaire, physical measurements, and provided biological samples, respectively.ConclusionsThe pilot study demonstrated the feasibility of recruitment and completion of the study protocols for the first large-scale cohort study designed to identify emerging risk factors for the major non-communicable diseases in the region.

Effect of the acute heat stress on serum endotoxin concentration and the expression of TLR4 mRNA in liver of Arbor Acres broiler chickens

This study was undertaken to investigate the relationship between gut-derived endotoxin and the Toll-like receptor 4 (TLR4) expression in the liver of broilers under acute heat stress (AHS). For this purpose, 120 Arbor Acres chicks were randomised into two groups: control temperature group (CT group, 22 ± 1°C) and high temperature group (HT group, 38 ± 1°C). The chicks received AHS at Day 28 and their small intestine, liver and blood samples were collected after 2 h, 5 h and 10 h to examine the histopathology, biochemical parameters, endotoxin concentrations and TLR4 expression. The results showed that damaged intestinal villi and severe congestion of the hepatic sinusoids were observed, especially after 10 h of AHS in the HT group. In addition, the levels of alanine transferase, aspartate transaminase, and direct bilirubin, except alkaline phosphataseafter were significantly elevated (P &lt; 0.05) and total bilirubin (P &lt; 0.01) and albumin (P &lt; 0.05) were decreased after 10 h of AHS as compared with the CT group, which are associated with liver function. Moreover, the mRNA expression of TLR4 in the liver was noticeably upregulated (P &lt; 0.05) during AHS with significantly increased in endotoxin concentration (P &lt; 0.01) of broilers. Altogether, these findings suggest that the upregulated expression of TLR4 mRNA was triggered via gut-derived endotoxin in heat stress-induced liver injury.

Integrated Lateral Flow Device for Flow Control with Blood Separation and Biosensing

Lateral flow devices are versatile and serve a wide variety of purposes, including medical, agricultural, environmental, and military applications. Yet, the most promising opportunities of these devices for diagnosis might reside in point-of-care (POC) applications. Disposable paper-based lateral flow strips have been of particular interest, because they utilize low-cost materials and do not require expensive fabrication instruments. However, there are constraints on tuning flow rates and immunoassays functionalization in papers, as well as technical challenges in sensors’ integration and concentration units for low-abundant molecular detection. In the present work, we demonstrated an integrated lateral flow device that applied the capillary forces with functionalized polymer-based microfluidics as a strategy to realize a portable, simplified, and self-powered lateral flow device (LFD). The polydimethylsiloxane (PDMS) surface was rendered hydrophilic via functionalization with different concentrations of Pluronic F127. Controlled flow is a key variable for immunoassay-based applications for providing enough time for protein binding to antibodies. The flow rate of the integrated LFD was regulated by the combination of multiple factors, including Pluronic F127 functionalized surface properties and surface treatments of microchannels, resistance of the integrated flow resistor, the dimensions of the microstructures and the spacing between them in the capillary pump, the contact angles, and viscosity of the fluids. Various plasma flow rates were regulated and achieved in the whole device. The LFD combined the ability to separate high quality plasma from human whole blood by using a highly asymmetric plasma separation membrane, and created controlled and steady fluid flow using capillary forces produced by the interfacial tensions. Biomarker immunoglobulin G (IgG) detection from plasma was demonstrated with a graphene nanoelectronic sensor integrated with the LFD. The developed LFD can be used as a flexible and versatile platform, and has the potential for detecting circulating biomarkers from whole blood. Sandwich-immunoassays can be performed directly on the LFD by patterning receptors for analytes on a desired substrate, and detections can be performed using a variety of sensing methods including nanoelectronic, colorimetric, or fluorescence sensors. The described bio-sensing technology presents an alternative for POC testing using small samples of human whole blood. It could benefit regions with limited access to healthcare, where delays in diagnosis can lead to quick deterioration of the quality of life and increase the morbidity and mortality.

A modified protocol for highly efficient EBV-mediated immortalization of human B lymphocytes from small volumes of peripheral blood serum

BackgroundMany human molecular and genetic studies require the use of a renewable biological material. Although primary fibroblast cell lines can be used for this purpose, there are disadvantages associated with human biopsies including the limited number of cell divisions. Peripheral blood has the advantage of being easier to obtain but also the drawback that blood cells produce only short-term cultures. Epstein-Barr virus is capable of transforming human B lymphocytes into indefinitely proliferating cells that can be maintained in tissue culture. Here, we report a convenient method of B-lymphocyte immortalization using small volumes of freshly collected human blood serum saturated with nucleated blood cells. Aim of the studyThe aim of the present study is modification and improvement of the protocol for highly efficient immortalization of human B lymphocytes from small volume of blood samples. Material and methodsCell line B95-8 was used as Epstein-Barr virus source for viral stock preparation. Immortalizing medium contains RPMI-1640, viral stock and additives. No feeder layer was used. ResultsAs result we present high efficient method for B lymphocytes immortalization with start blood volume less than 5 ml. ConclusionThe method is applicable for immortalization of B lymphocytes from small blood samples and is essential for studies involving children or patients when large blood volume sampling is impossible.

Serological survey of antibodies against Toxoplasma gondii in organic sheep and goat farms in Greece

Toxoplasmosis is a parasitic infection with global distribution caused by the protozoon Toxoplasma gondii. The present study is divided into the preliminary and the basic research. In the preliminary research, 413 serum samples (184 sheep and 229 goats) from organic farms of Péloponnèse and Western Sterea Hellas were tested for IgG antibodies against Τ gondii, by using enzyme-linked immunosorbent assay (ELISA). Blood sampling took place during spring of 2005 and samples were taken from male animals and female that had aborted during the reproductive period of 2004-2005. Significant prevalence of antibodies was the reason that further research of toxoplasmosis in organic farms was conducted. For the basic research, small ruminants' blood samples from organic farms of Péloponnèse, Western Sterea Hellas and the county of Ioannina, were collected during summer of 2006. Blood samples were collected from flocks with cases of abortions during the reproductive period of 2005-2006. Animals were separated into 3 groups: males, females that had abortions during the reproductive period of 2005-2006 and females that did not have abortions during the same period. A total number of 182 sheep sera and 167 goat sera were tested by using the enzyme-linked immunosorbent assay (ELISA). The results showed that antibodies were detected in 38.46% of male sheep, 60.87% of female sheep that had abortions during the reproductive period 2005-2006 and in 54.21% of female sheep that did not have abortions during the same period. Significant difference was determined only between male and female animals (p&lt;0.05). In addition, antibodies were detected in 12.12% of male goats, 14.29% of female goats that had abortions and in 22.35% of female goats that did not have abortions in the reproductive period 2005-2006. There was no significant difference between the results (p&gt;0.05). Seroprevalence in goats was significandy lower than that of the sheep (p&lt;0.05). In animals, aged less than 4 years old, seroprevalence was significantly lower (39.58% for sheep and 13.04% for goats) than that for animals aged more than 4 years old (61.97% for sheep and 24% for goats) (p&lt;0.05). The results of the survey suggest that there is high prevalence of toxoplasmosis both in animals that had aborted and in animals that had not aborted and that there are different species and gender susceptibility. Results also indicate that toxoplasmosis is more common in older animals.

1H-NMR metabolomic study of whole blood from hatchling loggerhead sea turtles (Caretta caretta) exposed to crude oil and/or Corexit.

We used proton nuclear magnetic resonance spectroscopy (1H-NMR) to evaluate metabolic impacts of environmentally relevant crude oil and Corexit exposures on the physiology of hatchling loggerhead sea turtles (Caretta caretta). Sample extraction and data acquisition methods for very small volume whole blood samples and sources of variation between individual hatchlings were assessed. Sixteen unclotted, whole blood samples were obtained from 7-day-old hatchlings after a 4-day cutaneous exposure to either control seawater, crude oil, Corexit 9500A or a combination of crude oil and Corexit 9500A. After extraction, one- and two-dimensional 1H-NMR spectra of the samples were obtained, and 17 metabolites were identified and confirmed in the whole blood spectra. Variation among samples due to the concentrations of metabolites 3-hydroxybutyrate, lactate, trimethylamine oxide and propylene glycol did not statistically correlate with treatment group. However, the characterization of the hatchling loggerhead whole blood metabolome provides a foundation for future metabolomic research with sea turtles and a basis for the study of tissues from exposed hatchling sea turtles.

Induced pluripotent stem cells (iPSC) from small volume peripheral blood samples for in vitro modeling and cell therapy of pediatric liver diseases

Aim: To define reprogramming and culture conditions allowing an effective and reproducible generation of high-quality iPSCs from small volume peripheral blood samples.

Theranos Experience Exposes Weaknesses in FDA Regulatory Discretion.

Theranos Experience Exposes Weaknesses in FDA Regulatory Discretion.

New method of sudomotor function measurement to detect microvascular disease and sweat gland nerve or unmyelinated C fiber dysfunction in adults with retinopathy

BackgroundDiabetes-associated microvascular complications such as retinopathy and neuropathy often lead to end-organ and tissue damage. Impaired skin microcirculation often precedes the detection of other advanced diabetic complications. The ANS-1 system contains a redesigned sympathetic skin response (ANS-1 SSR) device that measures sudomotor function, a photoplethysmography sensor, and a blood pressure device to comprehensively assess cardiac autonomic neuropathy and endothelial dysfunction. The purpose of this study was to determine the relationships between the ANS-1 SSR amplitude measured at the: (a) negative electrode (Nitric Oxide [NO] Sweat Peak) with microvascular diseases and associated vascular blood markers and (b) positive electrode (iSweat Peak) with C fiber function.MethodsAll participants (healthy controls n = 50 and retinopathy patients n = 50) completed the ANS-1 system evaluation and a basic sociodemographic and medical history questionnaire, including a quality of life measure (SF-36). A small sample of blood was drawn to determine levels of homocysteine, blood urea nitrogen (BUN), C-reactive protein (CRP), and fibrinogen. Symptoms of peripheral foot neuropathy were assessed with a scale from 1 (none) to 10 (the worst). We used Spearman rank correlations, independent samples t-tests, and receiver operating characteristic curves to determine the specificity and sensitivity of the NO Sweat Peak as a potential screening marker of retinopathy.ResultsThe ANS-1 System Cardiometabolic Risk Score and all indicators of quality of life on the SF-36, other than Emotional Role Functioning, were significantly worse in the retinopathy patients. The sudomotor response marker NO Sweat Peak had a sensitivity of 88% and a specificity of 68% (Area Under the Curve = 0.81, p < 0.0001) to detect retinopathy. The NO Sweat Peak response marker inversely correlated with BUN (ρ = −0.41, p < 0.0001), homocysteine (ρ = −0.44, p < 0.0001), fibrinogen (ρ = −0.41, p < 0.0001), the Cardiac Autonomic Neuropathy score (ρ = −0.68, p < 0.0001), and the heart rate variability Total Power (ρ = −0.57, p < 0.0001), and it positively correlated with the Photoplethysmography Index (PTGi; ρ = 0.53 p < 0.0001). The ANS-1 system sudomotor response marker iSweat Peak inversely correlated with the severity of symptoms on the peripheral neuropathy scale (ρ = −0.56, p < 0.0001).ConclusionThe results of the study show that this new method of measuring sympathetic skin response should be useful for detecting the earliest manifestations of microvascular disease and symptoms of C fiber dysfunction.

Repeatability and sources of variation of the bacteria‐killing assay in the common snapping turtle

Research on reptile ecoimmunology lags behind that on other vertebrates, despite the importance of such studies for conservation and evolution. Because the innate immune system is highly conserved across vertebrate lineages, assessments of its performance may be particularly useful in reptiles. The bacteria-killing assay requires a single, small blood sample and quantifies an individual's ability to kill microorganisms. The assay's construct validity and interpretability make it an attractive measure of innate immunity, but it requires proper optimization and sample storage. We optimized this assay for the common snapping turtle (Chelydra serpentina) to assess the repeatability of the assay and the effects of freezing and thawing on bactericidal capacity. We determined whether age (adult female and hatchlings) or incubation temperature influenced bactericidal capacity. We found that the assay was repeatable and that freezing plasma samples for 6 weeks at -80°C did not decrease bactericidal capacity nor did a single 30-min thaw and subsequent refreezing. However, we detected subtle interassay variation and results from one assay were 5-6% greater than those from the other two. Adult females had significantly greater bactericidal ability than hatchlings and we found no relationship between incubation temperature and bactericidal capacity. This assay is a useful tool in snapping turtles and may have applicability in other reptiles. However, species-specific optimization is required to ensure that variation among individuals exceeds interassay variation. Consideration should be given to optimization conditions that facilitate comparisons between or within groups, particularly groups that differ considerably in bactericidal capacity.