Alison Woodworth

Procalcitonin and C-reactive protein levels at admission as predictors of duration of acute brain dysfunction in critically ill patients

IntroductionNon-intensive care unit (ICU) cohorts have shown an association between inflammatory disturbances and delirium, though these relationships have not been studied in critically ill patients. This study sought to investigate the relationship between two inflammatory biomarkers, procalcitonin and C-reactive protein (CRP), and duration of acute brain dysfunction in ventilated patients.MethodsPatients enrolled in the Maximizing Efficacy of Targeted Sedation and Reducing Neurological Dysfunction (MENDS) trial were assessed daily for delirium using the Confusion Assessment Method-ICU. Plasma levels of procalcitonin and CRP were obtained within 24 hours of enrollment. Proportional odds logistic regression was used to examine the association between procalcitonin and CRP separately with delirium/coma-free days, adjusting for age, acute physiology score (APS) of the Acute Physiology And Chronic Health Evaluation (APACHE) II, sedation group (dexmedetomidine vs. lorazepam), and sepsis. Secondary analyses examined the association of these markers with other organ dysfunctions and 28-day survival.ResultsEighty-seven patients were included in this analysis. The median age of the patients was 60 years with APACHE II scores of 28; 68% had sepsis within 48 hours of admission. Higher levels of procalcitonin were associated with fewer delirium/coma-free days [odds ratio (OR), 0.5; 95% confidence interval (CI), 0.3 to 1.0; P = 0.04], whereas higher CRP levels showed trends towards fewer delirium/coma-free days (OR, 0.6; 95% CI, 0.3 to 1.1; P = 0.08). Similar relationships were found regardless of the presence of sepsis. No associations were found between procalcitonin or CRP with 28-day survival (P = 0.40 and 0.16, respectively).ConclusionsIn our pilot study, high baseline inflammatory biomarkers predicted prolonged periods of acute brain dysfunction, implicating inflammation as an important mechanism in the pathophysiology of delirium and coma during critical illness, irrespective of whether patients had sepsis or not.

Circulating MicroRNA miR-323-3p as a Biomarker of Ectopic Pregnancy

BACKGROUND The use of serum human chorionic gonadotropin (hCG) and progesterone to identify patients with ectopic pregnancy (EP) has been shown to have poor clinical utility. Pregnancy-associated circulating microRNAs (miRNAs) have been proposed as potential biomarkers for the diagnosis of pregnancy-associated complications. This proof-of-concept study examined the diagnostic accuracy of various miRNAs to detect EP in an emergency department (ED) setting. METHODS This study was a retrospective case-control analysis of 89 women who presented to the ED with vaginal bleeding and/or abdominal pain/cramping and received a diagnosis of viable intrauterine pregnancy (VIP), spontaneous abortion (SA), or EP. Serum hCG and progesterone concentrations were measured by immunoassays. The serum concentrations of miRNAs miR-323-3p, miR-517a, miR-519d, and miR-525-3p were measured with TaqMan real-time PCR. Statistical analysis was performed to determine the clinical utility of these biomarkers, both as single markers and as multimarker panels for EP. RESULTS Concentrations of serum hCG, progesterone, miR-517a, miR-519d, and miR-525-3p were significantly lower in EP and SA cases than in VIP cases (P < 0.01). In contrast, the concentration of miR-323-3p was significantly increased in EP cases, compared with SA and VIP cases (P < 0.01). As a single marker, miR-323-3p had the highest sensitivity of 37.0% (at a fixed specificity of 90%). In comparison, the combined panel of hCG, progesterone, and miR-323-3p yielded the highest sensitivity (77.8%, at a fixed specificity of 90%). A stepwise analysis that used hCG first, added progesterone, and then added miR-323-3p yielded a 96.3% sensitivity and a 72.6% specificity. CONCLUSIONS Pregnancy-associated miRNAs, especially miR-323-3p, added substantial diagnostic accuracy to a panel including hCG and progesterone for the diagnosis of EP.

False-Negative Results in Point-of-Care Qualitative Human Chorionic Gonadotropin (hCG) Devices Due to Excess hCGβ Core Fragment

BACKGROUND During pregnancy, human chorionic gonadotropin (hCG) immunoreactivity in urine consists of intact hCG as well as a number of hCG variants including the core fragment of hCGbeta (hCGbeta cf). We identified 3 urine specimens with apparent false-negative results using the OSOM(R) hCG Combo Test (Genzyme Diagnostics) qualitative hCG device and sought to determine whether an excess of 1 of the fragments or variants might be the cause of the interference. METHODS We measured concentrations of hCG variants in the urine from 3 patients with apparent false-negative hCG results. Purified hCG variants were added to urines positive for hCG and tested using the OSOM, ICON(R) 25 hCG (Beckman Coulter), and hCG Combo SP(R) Brand (Cardinal Health) devices. RESULTS Dilution of these 3 urine samples resulted in positive results on the OSOM device. Quantification of hCG variants in each of the 3 patient urine specimens demonstrated that hCGbeta cf occurred in molar excess of intact hCG. Addition of purified hCGbeta cf to hCG-positive urines caused false-negative hCG results using the OSOM and ICON qualitative urine hCG devices. CONCLUSIONS Increased concentrations of hCGbeta cf can cause false-negative results on the OSOM and ICON qualitative urine hCG devices.

Functional Expression of Arabidopsis thaliana Anthranilate Synthase Subunit I in Escherichia coli

Anthranilate synthase is involved in tryptophan (Trp) biosynthesis. Functional expression of subunit I from Arabidopsis (ASA1) was achieved in bacteria as a protein fused with glutathione S-transferase (GST). The active product was purified in a single step on a glutathione-Sepharose column. The Vmax (45 nmol min-1 mg-1), the apparent KM for chorismate (180 [mu]M), and the feedback inhibition by Trp (complete inhibition by10 [mu]M Trp) of the purified fusion product (GST-ASA1) were comparable to anthranilate synthase purified from plants. Polyclonal antibodies raised against the fusion protein product and purified by affinity chromatography on a GST-ASA1-Sepharose column cross-reacted with a 61.5-kD protein in a partially purified anthranilate synthase preparation from corn seedlings. GST-ASA1 cleavage by thrombin, as well as site-directed mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein.

Serum activin A does not predict ectopic pregnancy as a single measurement test, alone or as part of a multi-marker panel including progesterone and hCG.

BACKGROUND To evaluate the diagnostic accuracy of activin A alone or in a multi-marker panel for the prediction of ectopic pregnancy (EP). METHODS A retrospective analysis was performed on a cohort of 289 women who presented to the emergency department (ED) with vaginal bleeding and/or abdominal pain/cramping and were diagnosed with EP, spontaneous abortion, or viable intrauterine pregnancy. Serum progesterone, hCG, and activin A concentrations were measured on the samples obtained in the ED. Statistical analysis was performed to determine the clinical utility of these biomarkers as single measurement and as a multi-marker panel test for ectopic pregnancy. Women ≥18 y with vaginal bleeding or abdominal pain/cramping. RESULTS Progesterone (<10 ng/ml), hCG (<6,699 IU/l), and activin A (<0.26 ng/ml) cutoffs were optimized by ROC analysis. These demonstrated sensitivities of 62.9%, 74.2%, and 59.6%, and specificities of 60.5%, and 63.0%, and 61.0% respectively for detecting EP. The multi-marker panel utilizing all three biomarkers had a sensitivity of 70% and specificity of 69%. CONCLUSION Serum activin A cannot be used as a single measurement or in a multi-marker panel with progesterone and hCG to predict EP.

Molecular cloning and sequencing of cDNAs encoding insecticidal peptides from the primitive hunting spider, Plectreurys tristis (Simon).

Plectreurys tristis cephalothorax mRNA was isolated and amplified by PCR using degenerate primers corresponding to reverse translated mature Plt-VI toxin. An oligonucleotide corresponding to a portion of the amplified product was then used to screen a P. tristis cDNA library. The cDNAs from 10 positive clones were sequenced. Eight of these cDNAs corresponded to Plt-VI toxin, one to Plt-XI toxin, and one was very similar to Plt-VIII toxin, with the exception of a single amino acid substitution. Analysis of these cDNAs indicated that these toxins are initially synthesized as prepro-forms which undergo signal cleavage followed by additional processing at both their N- and C-termini to produce the mature products.

Analytical and clinical validation of the Immulite 1000 hCG assay for quantitative analysis in urine

BACKGROUND The Siemens Immulite hCG assay detects all major hCG variants in serum. Currently, this assay is only FDA approved for qualitative measurement of hCG in urine. METHODS Complete validation of the hCG assay in urine was performed on the Siemens Immulite 1000 immunoassay platform. Reference intervals were established for females <55 y, females ≥55 y, and males 20-70 y. RESULTS The limit of quantitation was 2.0 IU/l. The Immulite hCG assay was precise for measuring hCG in urine from pregnant patients with intra- and inter-assay imprecision of <11% CV. The assay was linear over a dynamic range of 2-2600 IU/l and 2-3500 IU/l for hCG and hCGβ respectively. The assay was non-linear for hCGβcf. No hook effect was observed at concentrations up to 1,200,000 pmol/l, for hCGβ or hCGβcf. The reference intervals were <2.0 IU/l for males, <2.2I U/l for females <55 y, and <12.2I U/l for females ≥55 y. CONCLUSION The Immulite 1000 hCG assay can accurately quantify hCG in urine.

Spurious elevation of serum PSA after curative treatment for prostate cancer: clinical consequences and the role of heterophilic antibodies

BACKGROUND: Various interferences can cause spurious results for common laboratory tests. Although rare, heterophilic antibodies may produce false elevations in PSA that could prompt unnecessary therapy in men previously treated for prostate cancer. The aim of this study was to determine the prevalence of small, spurious PSA elevations, and the role of heterophilic antibodies.METHODS: Phase I: all PSA tests drawn and measured between 27 October 2008 and 26 October 2010 at Vanderbilt University Medical Center were analyzed (n=17 133). Patients who had been treated for prostate cancer with PSA values that changed from undetectable to detectable were evaluated. Phase II: patients with a detectable PSA ⩽0.5 ng ml−1 measured between 24 October 2010 and 19 January 2011 were studied prospectively (n=1288). If any patient had a previously undetectable PSA value, their serum was tested for heterophilic antibody interference.RESULTS: Phase I: 11 men had a spuriously elevated PSA after curative treatment for prostate cancer (0.3%). Mean time to PSA elevation was 3.4±5.5 years, and mean elevation in PSA was 0.33±0.28 ng ml−1. Each patients PSA was undetectable after being repeated, and no patient went on to unnecessary treatment. Phase II: 10 men had a newly detectable PSA, 9 of whom had a history of prostate cancer. Each tested negative for interfering heterophilic antibodies when their PSA test was repeated with a heterophilic antibody-blocking reagent.CONCLUSIONS: In a large cohort, we estimate the prevalence of spuriously elevated PSA values in our population to be 0.3%. No patient with a prostate cancer history was subjected to unnecessary diagnostic evaluation or treatment. On prospective evaluation of PSA conversion to low detectable levels, no patient had evidence of interfering heterophilic antibodies. When using PSA for post-treatment surveillance, it is crucial to confirm all concerning values and consider the presence of a spurious elevation in PSA if the value does not correlate with the clinical scenario.

The Man/GaINAc-4-SO4-Receptor has Multiple Specificities and Functions

Carbohydrate-specific recognition by receptors contributes to a variety of different biological processes. These include: the folding and assembly of glycoproteins in the endoplasmic reticulum (Helenius 1994; Williams 1995; Trombetta and Helenius 1998), transport of glycoproteins to lysosomes (Kornfeld 1990, 1992), control of the circulatory half-life of individual glycoproteins (Drickamer 1991; Baenziger et al. 1992; Drickamer and Taylor 1993), endocytosis (Ashwell and Harford 1982; Drickamer and Taylor 1993), phagocytosis (Stahl and Ezekowitz 1998), cellular recognition during coagulation (McEver and Cummings 1997; Rosenerg et al. 1997), inflammation (Rosen and Bertozzi 1994; McEver et al. 1995; Lowe 1997), and development (Ioffe and Stanley 1994; Metzler et al. 1994; Chui et al. 1997). We recently described the first example of a carbohydrate-specific receptor, the Man/Ga1NAc-4-SO4 receptor (Fiete et al. 1998), that utilizes two distinct regions with different structural motifs to bind completely different carbohydrate structures. Furthermore, different cell types, such as macrophages and hepatic endothelial cells, express forms of the Man/Ga1NAc-4-SO4 receptor that differ with respect to their carbohydrate specificity. Macrophages express a mannose (Man) specific form of the receptor (Man-receptor), while hepatic endothelial cells express an N-acetylgalactosamine-4-SO4 (Ga1NAc-4-SO4) specific form of the receptor (Ga1NAc-4-SO4-receptor; Fiete and Baenziger 1997; Fiete et al. 1997). The regulated expression of different specificities by the Man/GalNAc-4-SO4-receptor indicates that the Man-receptor and the Ga1NAc-4-SO4receptor have different biological functions. Asparagine-linked carbohydrate structures terminating with the sequence SO4-4Ga1NAcβ1,4G1cNAcβ1,2Manα (S4GGnM) are present on highly restricted populations of glycoproteins, including the pituitary glycoprotein hormones LH and TSH (Green and Baenziger 1988a,b; Baenziger and Green 1991; Stockell Hartree and Renwick 1992; Thotakura and Blithe 1995). The synthesis of these sulfated oligosaccharides is protein specific, and is both hormonally and developmentally regulated (S.M. Dharmesh and J.U. Baenziger, unpubl. obs.; Dharmesh and Baenziger 1993; Manzella et al. 1997). Further, recognition of terminal Ga1NAc-4-SO4 on the oligosaccharides of LH by the Ga1NAc-4-SO4-receptor residing in hepatic endothelial cells (Fiete et al. 1991) plays a critical role in vivo by controlling the circulatory half-life of LH at key points during the ovulatory cycle (Baenziger et al. 1992; Baenziger 1996; Manzella et al. 1996). In contrast, recognition of carbohydrates terminating with Man, Fucose (Fuc), or N-acetylglucosamine (G1cNAc) by the Man-receptor, present on terminally differentiated macrophages, is thought to be essential for phagocytosis of pathogens such as yeasts, bacteria, and viruses (Fraser et al. 1998; Stahl and Ezekowitz 1998). Thus, the Man/Ga1NAc-4-SO4-receptor, in the form of the Man-receptor or the GalNAc-4-SO4-receptor, may fulfill distinct roles that reflect its ligand specificity as well as the setting in which it is expressed.

Pragmatic pharmacology: population pharmacokinetic analysis of fentanyl using remnant samples from children after cardiac surgery.

AIMS One barrier contributing to the lack of pharmacokinetic (PK) data in paediatric populations is the need for serial sampling. Analysis of clinically obtained specimens and data may overcome this barrier. To add evidence for the feasibility of this approach, we sought to determine PK parameters for fentanyl in children after cardiac surgery using specimens and data generated in the course of clinical care, without collecting additional blood samples. METHODS We measured fentanyl concentrations in plasma from leftover clinically-obtained specimens in 130 paediatric cardiac surgery patients and successfully generated a PK dataset using drug dosing data extracted from electronic medical records. Using a population PK approach, we estimated PK parameters for this population, assessed model goodness-of-fit and internal model validation, and performed subset data analyses. Through simulation studies, we compared predicted fentanyl concentrations using model-driven weight-adjusted per kg vs. fixed per kg fentanyl dosing. RESULTS Fentanyl clearance for a 6.4 kg child, the median weight in our cohort, is 5.7 l h(-1) (2.2-9.2 l h(-1) ), similar to values found in prior formal PK studies. Model assessment and subset analyses indicated the model adequately fit the data. Of the covariates studied, only weight significantly impacted fentanyl kinetics, but substantial inter-individual variability remained. In simulation studies, model-driven weight-adjusted per kg fentanyl dosing led to more consistent therapeutic fentanyl concentrations than fixed per kg dosing. CONCLUSIONS We show here that population PK modelling using sparse remnant samples and electronic medical records data provides a powerful tool for assessment of drug kinetics and generation of individualized dosing regimens.