Use of high-sensitivity digital ELISA improves the diagnostic performance of circulating brain-specific proteins for detection of traumatic brain injury during triage

ABSTRACT

Background: Historically, limited sensitivity associated with traditional immunoassay methods has prevented the use of brain-specific proteins as blood biomarkers of traumatic brain injury (TBI) during triage, as these proteins exhibit low circulating concentrations. Digital ELISA is a newly-developed technique that is up to 1000 times more sensitive than conventional ELISA methods. The purpose of this study was to determine whether the use of digital ELISA over conventional ELISA improves the performance of brain-specific proteins as blood biomarkers of TBI during triage.

Methods: Blood was sampled from TBI patients (n = 13) at emergency department admission, as well as from neurologically normal controls (n = 72). Serum levels of two brain-specific proteins, neurofilament light chain (NfL) and Tau, were measured via digital ELISA. Estimated conventional ELISA measures were generated by adjusting values according to the lower limits of detection achievable with commercially available conventional ELISA assays, and receiver operating characteristic (ROC) analysis was used to compare the diagnostic performance of digital ELISA measures to estimated conventional ELISA measures in terms of their ability to discriminate between TBI patients and controls.

Results: Used in combination, digital ELISA measures of NfL and Tau could discriminate between groups with 100% sensitivity and 91.7% specificity. Estimated conventional ELISA measures could only discriminate between groups with 7.7% sensitivity and 94.4% specificity. This difference in diagnostic performance was statistically significant when comparing areas under ROC curves.

Conclusions: The use of digital ELISA over conventional ELISA methods improves the diagnostic performance of circulating brain-specific proteins for detection of TBI during triage.     

Genetic variation in alcohol dehydrogenase is associated with neurocognition in men with HIV and history of alcohol use disorder: preliminary findings

The co-occurrence of HIV and alcohol use disorder (AUD) amplifies risk for neural injury and neurocognitive deficits. However, the substantial neurocognitive heterogeneity across HIV+/AUD+ individuals suggests inter-individual differences in vulnerability to the neurotoxicity of comorbid HIV/AUD. Genetic variation in alcohol dehydrogenase (ADH), which metabolizes ethanol, may contribute to inter-individual neurocognitive variability. We evaluated associations between five ADH single-nucleotide polymorphisms (SNPs) and neurocognition in men stratified by HIV and lifetime AUD status. Neurobehavioral assessments were administered to 153 men. Three-way ANOVAs examined the interaction of HIV, AUD, and ADH SNPs on global and domain-specific demographically corrected T scores. Follow-up ANCOVAs adjusted for age, estimated verbal IQ, depression, and remote non-alcohol substance use disorders. HIV/AUD groups differed globally and for verbal fluency, working memory, executive function, and processing speed T scores specifically, with HIV+/AUD+ exhibiting the poorest performance. ADH4 (rs1126671) was associated with large effects on working memory (d?=???1.16, p?=?.001) and executive function (d?=???0.77, p?=?.028) selectively in HIV+/AUD+, which remained significant in ANCOVA models. ADH1A (rs3819197) moderated the deleterious effects of HIV+/AUD+ on processing speed such that HIV+/AUD+ related to slower information processing in A allele carriers but not GG homozygotes (ps?<?0.03). Preliminary findings suggest genetic variation in the ADH pathway moderates the deleterious neurocognitive effects of comorbid HIV/AUD. Differential metabolism of heavy ethanol exposure may compromise neurocognition under conditions of neurobiological stress, such as in HIV infection. The functional effects on ethanol metabolism of ADH SNPs examined in this study remain poorly understood, warranting further examination of pharmacokinetic mechanisms mediating ADH gene-neurobehavior relationships in HIV.

     

In-vitro recombination in rad and rnc mutants of Saccharomyces cerevisiae

Summary Extracts of S. cerevisiae cells can catalyze homologous recombination between plasmids in vitro. Extracts prepared from rad50, rad52 or rad54 disruption mutants all have reduced recombinational activity compared to wild-type. The rad52 and rad54 extracts are more impaired in the recombination of plasmids containing double-strand breaks than of intact plasmids, whereas rad50 extracts are deficient equally for both types of substrate. The nuclease RhoNuc (previously designated yNucR), encoded by the RNC1 (previously designated NUC2) gene and regulated by the RAD52 gene, is not required for recombination when one substrate is single-stranded but is essential for the majority of recombination events when both substrates are double-stranded. Furthermore, elimination of this nuclease restores recombination in rad52 extracts to levels comparable to those in wild-type extracts.     

Utilization review of the use of BACTEC PLUS high-volume blood culture bottles.

The BACTEC PLUS 26 (NR26) (Becton Dickinson, Towson, Md.) high-volume blood culture bottle replaced the less expensive smaller-volume NR6A bottle in our hospital. An audit carried out several months after their introduction revealed that only 17.5% of the NR26 bottles received the required blood volume. Several audits and educational programs were required in order to achieve a compliance rate of > 60%.     

Cross-reactive idiotopes among anti-foot and mouth disease virus neutralizing antibodies.

Foot and mouth disease virus (FMDV) viral protein 1 is the only one of the four viral proteins (VP) that induces neutralizing antibodies as an isolated protein. A 32 amino acid (AA) residue (32dimer) of FMDV subtype A12 Lp ab VP1 (AA 137-168) was immunogenic against the A12 subtype. Three antibody populations each recognizing different epitopes on 32dimer were isolated by affinity chromatography (AFC) from the serum of a steer which had been immunized with the 32dimer. The 32dimer contains an AA sequence that is recognized by a protective paratope carried on a murine monoclonal antibody (mAb) (7SF-3.H3.1). Polyclonal anti-7SF-3 idiotype antibodies specifically inhibited the binding activity of one of these anti-32dimer antibody populations suggesting the existence of cross-reactive paratopic-related idiotopes between mAb 7SF-3 and antibodies elicited by the 32dimer. These anti-idiotypic antibodies were used in AFC to purify antibodies from the anti-32dimer serum. The purified antibody population has characteristics that resemble those of the mAb 7SF-3, i.e. its reactivity with FMDV A subtypes in ELISA, radioimmunoassay (RIA), mouse neutralization and its lack of reactivity with a mAb 7SF-3 neutralizing escape virus variant. Furthermore, these antibodies were specifically inhibited by either anti-mAb 7SF-3 idiotypic antibodies or peptides containing the mAb 7SF-3 epitope. Using the same experimental approach, mAb 7SF-3 idiotope-bearing antibodies were shown to be present in serum from bovine and swine convalescent from FMDV A12 Lp ab infection. Thus, the highly immunogenic area between residues 137 and 168 of FMDV VP1 elicited a cross-reactive neutralizing idiotope response conserved amongst several animal species.     

Interstitial Duplication of 7(q22→q34)

We report on a 3-year-old boy with an interstitial duplication of 7(q22→q34), confirmed with fluorescent in-situ hybridization. He had post-natal growth retardation, developmental delay, frontal and parietal bossing, deep-set eyes, strabismus, bilateral optic nerve hypoplasia, and mild dilatation of the cerebral ventricles. His Phenotype was not significantly different from that of the three previously reported patients with interstitial duplication of the smaller segment 7(q22→q31). © 1993 Wiley-Liss, Inc.     

Calpain proteolysis of von Willebrand factor enhances its binding to platelet membrane glycoprotein IIb/IIIa: an explanation for platelet aggregation in thrombotic thrombocytopenic purpura

We have previously observed calpain activity (calcium-dependent cysteine protease) in sera from patients with acute thrombotic thrombocytopenic purpura (TTP). The calpain activity was not present following recovery and was not detected in other thrombocytopenic disorders. We postulated that this enzyme could participate in the pathogenesis of TTP. Because other investigators have demonstrated abnormalities of von Willebrand factor (vWF) in patients with TTP, we proposed that calpain might interact with vWF in TTP. To challenge this hypothesis, we measured the binding of untreated and calpain-treated vWF to normal and ADP or calpain activated platelets. Untreated vWF bound in a specific and saturable fashion to activated platelets, but only at low (30 microM) calcium concentrations. Von Willebrand factor did not bind to activated platelets at physiological (2 mM) calcium concentrations. Calpain proteolysis of vWF changed the binding characteristics of the vWF so that it had greatly increased binding to both ADP and calpain activated platelets. The calpain-proteolyzed vWF bound to activated platelets at both low and physiological calcium concentrations, and was capable of causing platelet aggregation. The calpain-proteolyzed vWF bound to the activated platelets via glycoproteins IIb/IIIa as demonstrated by inhibition studies using monoclonal antibodies against glycoproteins IIb/IIIa and Ib. It also had a high binding affinity and was capable of inhibiting the binding of radiolabelled fibrinogen to the activated platelets at physiological calcium concentrations. Calpain also proteolyzed fibrinogen, but the calpain altered fibrinogen had normal platelet reactivity. These studies provide further insight into the pathogenesis of the platelet aggregation of thrombotic thrombocytopenic purpura. Calpain proteolyses vWF and can produce the characteristic loss of large multimers seen on sodium dodecyl sulphate (SDS)-agarose gel electrophoresis. The altered vWF is highly reactive with activated platelets and binds to platelet glycoproteins IIb/IIIa and participates in formation of the platelet aggregates that characterize this disease.     

Endotoxin-induced tumor necrosis factor activity production by equine peritoneal macrophages

A study was performed to determine whether equine macrophages produce tumor necrosis factor (TNF) activity in vitro in response to endotoxin and to study the effects of endotoxin concentration and incubation time on the amount of TNF produced. Equine peritoneal macrophages were isolated and cultured in vitro for 2, 6, 12, or 24 hr in tissue culture media containing 1) no additive (nonstimulated control), 2) endotoxin (0.5 ng/ml, 5 ng/ml, or 5 micrograms/ml), or 3) the calcium ionophore A23187 (0.95 microM). The supernatant media concentrations of TNF activity were determined by an in vitro cytotoxicity bioassay using the murine fibrosarcoma cell line WEHI 164 clone 13. At all incubation times, macrophages exposed to endotoxin produced significantly more TNF than did nonstimulated control cells (P less than 0.05). Among the three doses of endotoxin tested, there were no differences in concentrations of TNF activity in the supernatant media. The mean concentration of TNF activity in media from nonstimulated macrophages was significantly less after 24 hr incubation than after 2, 6 or 12 hr. Incubation time had no effect on TNF production by macrophages exposed to 0.5 ng/ml and 5 micrograms/ml endotoxin, but macrophages exposed to 5 ng/ml endotoxin produced significantly more TNF after 6 and 12 hr than after 2 hr. To address the reason for the decrease in TNF activity in media from nonstimulated macrophages over time, a second study was designed to determine the effect of incubation on the activity of TNF under tissue culture conditions. Human recombinant TNF was added to media that was incubated 2-24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Single, total paracentesis for tense ascites: sequential hemodynamic changes and right atrial size

Hemodynamic changes induced by a single, total paracentesis were evaluated in 21 patients with tense ascites from whom 4 to 16 L of ascites were drained over 2 to 8 hr with no serious complications. At 60 min, compared to baseline, there was an increase in cardiac output (7.7 +/- 0.5 to 8.5 +/- 0.6 L/min, p less than 0.02) and a tendency for right atrial pressure to decrease (9.3 +/- 0.8 to 7.50 +/- 0.8 mm Hg, NS), with no change in pulmonary capillary wedge pressure (10.9 +/- 0.9 to 10.7 +/- 0.9 mm Hg). Between 3 and 12 hr later, there was a drop in right atrial pressure, pulmonary capillary wedge pressure and cardiac output to 5.6 +/- 0.6 (p less than 0.02), 7.2 +/- 0.8 mm Hg (p less than 0.002) and 7.2 +/- 0.6 L/min (NS) respectively, indicative of the development of relative hypovolemia and suggesting that therapeutic plasma expansion is appropriate at this time. Two-dimensional echocardiography before paracentesis (n = 8) showed a reduction in the right to left atrium area ratio as compared with values in patients with minimal ascites (0.54 +/- 0.04 vs 0.82 +/- 0.02, p less than 0.0001). This technique may help in identifying patients with right atrial compression caused by tense ascites.     

Occupational hazards to health care workers: diverse, ill-defined, and not fully appreciated

Health care workers are challenged by an imposing group of occupational hazards. These hazards include exposure to ionizing radiation, stress, injury, infectious agents, and chemicals. The magnitude and diversity of these hazards are not fully appreciated. The acquired immunodeficiency syndrome epidemic has created additional occupational hazards and has focused attention on the problem of occupational hazards to health care workers. Concern over the nosocomial transmission of the human immunodeficiency virus has contributed to efforts to implement universal infection control precautions and to decrease needlestick injuries. Health care organizations and providers, who have prompted health and safety campaigns for the general public, should not overlook the dangers associated with the health care setting.