Neutrophils May Predict Development of Sepsis

The movement of white blood cells may predict burn patients who are at risk for sepsis, days before diagnosis is currently possible, according to a study published in PLOS ONE on Dec. 9, 2014. Using a novel microfluidic device, the researchers discovered that neutrophils from burn patients who went on to develop sepsis spontaneously migrate in the absence of chemical attractants, while neutrophils from healthy patients and burn patients without sepsis don't move. The researchers hope that with further validation and refinement, a microfluidic test that assesses neutrophil movement can be used to predict sepsis and monitor effectiveness of antibiotic therapy. While the current study was conducted on burn patients, the researchers believe evaluation of neutrophil movement may be useful in other patients at risk for sepsis. “The most common blood test ordered to evaluate a patient’s ability to fight infection is absolute neutrophil count, based on the assumption that — like well-trained soldiers — neutrophils are always fast, disciplined and effective in pursuing their targets, meaning that the size of the neutrophil ‘army’ is all that matters," said co-author Daniel Irimia, M.D., Ph.D., in a statement. "Our work challenges that assumption and shows that, even when the number of neutrophils is unchanged, the army can fall into disarray and become ineffective.” Neutrophils comprise the majority of white blood cells involved in immune responses. They travel to infection sites in response to chemical signals. The authors say that earlier efforts to characterize neutrophil chemotaxis in sepsis (such as the transwell assay and early microfluidic assays) were limited by the instability of the chemical gradients and the lack of single cell resolution. The researchers designed microfluidic devices to quantify the neutrophil migration phenotype with high precision. The narrow channels included straight sections to measure the speed and persistence of the cells’ motion, as well as branches and obstacles to test the cells’ ability to make directional decisions. Isolated neutrophils were loaded into the device and assessed in three distinct conditions: serum-free Hank's Balanced Salt Solution and gradients of formyl-methionyl-leucyl-phenylalanine and leukotriene B4 to test a total of 18 independent parameters. The device quantified neutrophil migration from 74 blood samples (taken from 13 patients with major burns and three healthy subjects) for more than four weeks. The researchers found that neutrophils from healthy individuals moved "quickly and efficiently" toward a chemical attractant, while cells from burn patients (regardless of subsequent sepsis) showed "limited, slower, and poorly organized movement." Additionally, the researchers found "spontaneous migration" of neutrophils in the absence of a chemo-attractant in burn patients with sepsis. This spontaneous migration was witnessed before sepsis diagnosis and disappeared after sepsis was effectively treated. The spontaneous neutrophil migration phenotype was "rare" in patients with major burns in the absence of sepsis and was not seen in healthy individuals. "Overall, sensitivity and specificity values for our assay are only slightly better than other markers for sepsis in burn patients. However, it is important to note that, enabled by technology, we could observe the changes in neutrophil migration phenotype, up to two days before sepsis," explains Irimia. The analysis of neutrophil migration phenotype can be completed in less than 4 hours after the blood samples become available. Irimia says the next major step in the research is to test, this year, the applicability of the findings to prediction of sepsis in other patients with critical conditions. Additionally, he tells DTET that future iterations of the microfluidic device, which doesn't require a full laboratory, will address streamlining the blood sample processing protocols before neutrophils are loaded, making the device more user friendly, automating the image analysis. Takeaway: Microfluidic evaluation of neutrophil migration may play a role in earlier diagnosis of sepsis and monitoring of the effectiveness of antibiotic treatment. While the current study was conducted in samples from major burn patients, the researchers believe neutrophil migration may be applicable to other critically ill patients as well. Urine Test Can Tailor Kids' Asthma Therapy Dosage A new urine test can help personalize anti-inflammatory treatment for pediatric asthma patients, according to an abstract presented at the British Thoracic Society Winter Meeting (England; Dec. 3–5, 2014). While further validation is needed, the authors say that a urine prostaglandin can predict future risk of asthma exacerbation better than other asthma assessment measures including sputum eosinophils, the childhood asthma control test (C-ACT), or spirometry. Prostaglandin metabolites are released as part of an inflammation reaction by immune cells in the airway lining, following exposure to an asthma trigger. Noninvasive measurement of this airway inflammation can improve treatment decisions, including dosing of anti-inflammatory therapies. Urine samples were prospectively compared between non-asthmatic children (n=48) and asthmatic children (n=25; aged 7 years to 15 years) on days when they showed no symptoms. Additionally, the researchers assessed asthma exacerbations (measured as the number of days of receipt of unscheduled medical attention or missed school due to asthma symptoms). Urine prostaglandin metabolites (PGD, PGE and PGJ) were measured using high-performance liquid chromatography–mass spectrometry (HPLC-MS). Sampling was repeated at three months. Compared to controls, urine metabolites PGD₂, PGE₂ and PGJ₂ were increased in asthma patients. Low levels of the protective prostaglandin metabolite 15-dPGJ₂ significantly predicted subsequent asthma exacerbation within three months (positive predictive value, 75 percent; negative predictive value, 90 percent). Sputum eosinophil measurement, spirometry, and C-ACT did not predict subsequent exacerbations. "When children see their general practitioner for their annual review, we hope that this test can help indicate the level of steroid medication they actually need," said lead author Rossa Brugha from Queen Mary University of London (United Kingdom) in a statement. "If implemented it will help the child to manage their asthma more effectively and hopefully reduce the number of asthma attacks.” Brugha tells DTET that validation is needed in a larger prospective trial and that the group is awaiting funding to complete that. Additionally, he says that they hope to make the test easier to perform. Takeaway: A noninvasive urine test may aid clinicians in personalizing dosage of anti-inflammatory treatments for asthmatic children. RNA in Sputum May Improve CT's Diagnosis of Lung Cancer Micro RNA (miRNA) markers found in sputum may improve the accuracy of lung cancer diagnosis in conjunction with low-dose CT (LDCT) screening, according to a study published Jan. 15 in Clinical Cancer Research. The growing use of LDCT as a lung cancer-screening tool among smokers has been accompanied by a "tremendous" rise in the number of lung nodules detected, many of which are benign. The authors say clinical use of a future iteration of this noninvasive RNA-panel test may "dramatically" decrease the need for follow-up imaging and biopsies. "The higher positive predictive value (PPV; 84 percent) of the biomarkers as compared with only two percent PPV of LDCT indicates that the biomarkers would result in much less overdiagnosis," writes senior author Feng Jiang, M.D., Ph.D., from University of Maryland, Baltimore. "The positive cases detected by the biomarkers in CT-found solitary pulmonary nodules (SNPs) are malignant SPNs, and should have instant surgical treatment. Furthermore, the negative cases discovered by the biomarkers in CT-found SPNs are benign growths, and will not be followed up." It is estimated that one-fourth of all identified nodules are indeterminate based on CT, but that the vast majority (greater than 96 percent) ultimately prove to be false positives. But in making that determination, patients are subjected to ongoing, costly imaging and related radiation exposure, as well as invasive biopsies. The researchers initially evaluated expression of 13 sputum miRNAs previously identified as signatures of lung cancer using quantitative reverse transcriptase polymerase chain reaction. The training set consisted of 122 patients (60 with malignant SPNs and 62 with benign SPNs). While all 13 miRNAs differed significantly between patients with lung cancer and those with benign nodules, miRs-21, 31, and 210 were selected as the best markers. The 3-marker panel was then validated in an internal testing set of 136 patients (67 malignant and 69 benign) and in an external testing cohort of 155 patients (76 malignant and 79 benign). The three miRNA markers could diagnose early-stage lung cancer among SPNs with 81 percent to 83 percent sensitivity and 86 percent to 88 percent specificity, yielding a PPV of 84 percent and a negative predictive value of 81 percent. The authors concede, though, that the accuracy of this panel is "not high enough" for clinical use, so ongoing efforts are aimed at identifying additional miRNA markers to expand the panel. The three miRNAs could not differentiate between stage I and stage II disease and the markers were not affected by age, gender, or ethnicity. Takeaway: miRNAs offer hope as a noninvasive means to improve the diagnostic accuracy of LDCT lung cancer screening and ultimately reduce the need for follow-up imaging and invasive biopsies to determine indeterminate nodules' pathogenicity. Panel Could Eliminate Need for Surgical Diagnosis of Endometriosis Patterns of genetic activity can be used to diagnose and stage endometriosis, according to a study published in the December 2014 issue of Endocrinology. This could represent a significant breakthrough in providing earlier, less invasive diagnosis of endometriosis, which is estimated to affect 10 percent of all reproductive-age women. Endometriosis is an inflammatory disorder that affects 50 percent of women with pelvic pain and/or infertility (estimated to be more than 100 million women worldwide). Definitive diagnosis of the condition is typically prolonged for up to a decade because it requires surgery and presents with nonspecific symptoms. "A prompt, low-risk, low-cost diagnostic with high accuracy is needed to shorten time to diagnosis, minimize disease progression and ovarian cancer risk, optimize timing and strategies for pain and infertility therapies, and monitor disease recurrence," writes coauthor Linda Giudice, M.D., Ph.D., from University of California, San Francisco. The researchers identified classifiers using genomic data from 148 archived endometrial samples from women (aged 20 to 50 years old) with (n = 77) or without endometriosis (normal controls, n = 34; or with other common uterine/pelvic pathologies, n = 37) across the menstrual cycle. The performance of the classifiers was then evaluated in independent sample sets. Ninety-two differentially expressed genes were analyzed using microarray technology and machine learning algorithms were used to develop a grouping system to analyze the gene activity of endometrium tissue samples. The best performing classifiers, the researchers report, identified endometriosis with 90 percent to 100 percent accuracy and were menstrual cycle phase-specific or independent. The classifiers could additionally distinguish between samples from endometriosis patients and those patients with other uterine disorders, could differentiate between endometriosis stages, and could identify endometriosis at different points in the menstrual cycle. Takeaway: A microarray panel consisting of protein and gene markers may lead to less invasive and earlier diagnosis of endometriosis.