Small Amount Of Blood Research Articles

On the Occurrence of a Haemogregarinae (Apicomplexa) Parasite from Freshwater Turtles of South 24 Parganas, West Bengal, India

The aim of this study was to investigate Haemogregarine parasites (Apicomplexa: Haemogregarinidae) of freshwater turtles (Lissemys punctata andersoni, Geoclemys hamiltonii) of India. Turtles were collected by net from two ponds of South 24 Parganas, West Bengal, India. A small amount of blood was taken from the subcarapacial vein puncture site. The blood smears were prepared and air dried and fixed in absolute methyl alcohol. The slides were stained with Giemsa. Haemogregarine parasites were recorded from the erythrocytes of turtles. Multiple stages of intraerythrocytic gametocytes (microgametocytes, macrogametocytes, early schizonts and mature schizonts) were observed in blood films. It was found that only twenty out of the twenty five turtles (80%) were infected with the parasite. The prevalence rate was higher in larger turtles in comparison to smaller ones. It was also found that female turtles had a higher prevalence of infection than males.

An ultrasound model to calculate the brain blood outflow through collateral vessels: a pilot study

BackgroundThe quantification of the flow returning from the head through the cervical veins and the collaterals of the internal jugular vein (IJV), is becoming of prominent interest in clinical practice. We developed a novel model to calculate the cerebral venous return, normalized to the arterial inflow, in the different segments of the IJV.MethodsWe assessed, by established Echo Colour Doppler (ECD) methodology, the head inflow (HBinF) defined as the sum of common carotids and vertebral arteries, as well as the cerebral flow (CBF) defined as the sum of internal carotid and vertebral arteries. We also assessed the head outflow (HBoutF) defined as the sum of the measurements at the junction of the IJV and the vertebral veins. In addition, we also calculated the collateral flow index (CFI) by estimating the flow which re-enters directly into the superior vena cava as the amount of blood extrapolated by the difference between the HBinF and the HBoutF. We preliminarily tested the model by comparing ten healthy controls (HC) with ten patients affected by chronic cerebral spinal venous insufficiency (CCSVI), a condition characterized by some blockages in the IJV which are bypassed by collateral circulation.ResultsIn HC the HBinF was 956+-105ml/min, whereas the HBoutF was > 90% of the HBinF, leading to a final CFI value of 1%. The last result shows that a very small amount of blood is drained by the collaterals. In upright we confirmed a reduction of the outflow through the IJV which increased CFI to 9%. When we applied the model to CCSVI, the HBinF was not significantly different from controls. In supine, the flow of CCSVI patients in the IJV junction was significantly lower (p < 0.001) while the correspondent CFI value significantly increased (61%, p < 0.0002).ConclusionsOur preliminary application of the novel model in the clinical setting suggests the pivotal role of the collateral network in draining the blood into the superior vena cava under CCSVI condition.

Index of Suspicion in the Nursery

A 2-week-old Hispanic male infant is admitted with a 1-day history of subjective fever, nonbilious vomiting, decreased oral intake, and lethargy. The physical examination is normal other than two small pustules, one on the cheek and the other on the scalp; both are sent for culture. He was born at 33 5/7 weeks’ gestation to a 27-year-old G8-P6-5-1-1-6 woman. The mother had prenatal care starting at approximately 4 months of gestation that was complicated by premature and prolonged rupture of membranes (22 hours) with purulent amniotic fluid. Her laboratory evaluation included the following: blood group AB positive, rapid plasma reagin nonreactive, hepatitis B negative, HIV negative, and urine drug screen negative. Her group B streptococcus, chlamydia, gonorrhea, and rubella screens are unknown. The mother received azithromycin and metronidazole during labor and delivery. The infant was delivered via emergent cesarean delivery for chorioamnionitis. His Apgar scores were 4 and 8 at 1 and 5 minutes, respectively. The infant had neither a feeding problem nor apnea/bradycardia and received ampicillin and gentamicin intravenously for 7 days due to the maternal chorioamnionitis. His blood culture was negative. He was discharged home at 9 days of age. When he arrives back at 2 weeks of age, a complete evaluation for neonatal sepsis is initiated and herpes simplex virus infection is suspected. He receives empiric treatment with acyclovir, vancomycin, and cefotaxime. ### Admission Laboratory Data CBC: white blood cell (WBC) counts 6,400/mm3; Hgb, 13.7 g/dL; Hct, 40.9%; and platelets, 409,000/mm3. WBC counts differential: segmented forms, 55%; bands, 4%; lymphocytes, 26%; and monocytes, 14%. Urinalysis: specific gravity, 1.015; negative glucose, small amount of blood, trace protein, no WBCs/high power field, 0 to 2 red blood cells/high power field. CSF: glucose, 34 mg/dL; protein greater than 460 mg/dL; red blood cell count, 26,200/mm3; WBC count, 100/mm …

Development of a stable isotope dilution UPLC‐MS/MS method for quantification of dexmedetomidine in a small amount of human plasma

Dexmedetomidine (Dex) is a selective central α2-agonist with anesthetic properties and has been used in clinical practice for sedation in the intensive care unit (ICU) after operations. In this study, an analytical assay for the determination of Dex in a small amount of plasma was developed for the application to pediatric ICU trials. The quantification of Dex was constructed using the original stable isotope Dex-d3 for electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in the selected reaction monitoring mode. A rapid ultra-performance liquid chromatography technique was adopted using ESI-MS/MS with a runtime of 3 min. Efficacious concentration levels (50 pg/mL to 5 ng/mL) could be evaluated using a very small amount of plasma (10 μL) from patients. The lower limit of the quantification was 5 pg/mL in the plasma (100 µL). For sample preparation, a solid-phase extraction was used along with the OASIS-HLB cartridge type. Recovery values ranged from 98.8 to 100.3% for the intra- [relative standard deviation (RSD), 0.9-1.3%] and inter- (RSD, 0.9-1.5%) day assays. A stable test had recovery values that ranged from 97.8 to 99.7% with an RSD of 1.0-1.9% for the process/wet extract, bench-top, freeze-thaw and long-term tests. This method was used to measure the Dex levels in plasma from pediatric ICU patients. In the clinical ICU trial, the small amount of blood (approximate plasma volume, 200 μL) remaining from blood gas analysis was reused and targeted for the clinical analysis of Dex in plasma.

Comparison of a Rapid Bed-Side Test with a Central Laboratory Analysis for C-Reactive Protein in Newborn Infants with Suspicion of Sepsis

Neonatal sepsis is one of the leading causes of neonatal morbidity and mortality in the newborn period. The purpose of this study was to compare a rapid bed-side test against central laboratory analysis for CRP in newborn infants with suspicion of sepsis. We included 23 newborn infants with suspected or proven sepsis (group 1). Age and gender matched 40 infants without sepsis were assigned as controls (group 2). A total of 116 blood samples (58 samples from each group) were drawn from the peripheral vein for C-reactive protein (CRP) measurements. For the NycoCard CRP tests, 5 microL of whole blood and the same amount of serum was used to determine the CRP value (wCRP-N and sCRP-N). As the reference CRP test, serum concentrations of CRP (CRP-Lab) were simultaneously measured by immunonephelometric method requiring 1 mL of serum sample. CRP values > or = 10 mg/L were considered positive for all three tests. It was found that the median values of wCRP-N (18.9 mg/L), sCRP-N (25 mg/L), and CRP-Lab (22.2 mg/L) of group 1 were higher than group 2 (wCRP-N: 1.1 mg/L, sCRP-N: 5 mg/L, and CRP-Lab: 2.9 mg/L) (p < 0.001). Nycocard-CRP were positively correlated with CRP-Lab. With considering CRP-Lab positive at > 10 mg/L, both CRP-NycoCard tests have high sensitivity and specificity, although sCRP-N has slightly higher predictive values. Passing-Bablok regression and Bland-Altman analysis showed that NycoCard tests have no statistically significant systematic proportional bias. Nycocard-CRP tests have high predictive values, require a very small amount of blood, and results can be obtained quickly. Determinations of CRP concentrations by NycoCard-CRP test seems to be helpful in distinguishing septic and aseptic newborns.

Four-part leukocyte differential count based on sheathless microflow cytometer and fluorescent dye assay

Leukocyte differential count is one of the most frequently ordered clinical tests in hospitals. This paper reports a point-of-care test for the leukocyte count by using a microflow cytometer and a fluorescent dye assay. The dye assay relied on fluorescent detection alone to count leukocytes in blood and to identify leukocyte subtypes. By combining the fluorescent assay with a sheathless microflow design, the proposed method achieved a minimal sample volume by eliminating excessive dilution and sheath flow. In this paper, a four-part leukocyte differential count including lymphocyte, monocyte, neutrophil and eosinophil was demonstrated, and the whole test consumed only a small amount of blood (5 μL) and reagents (68 μL in total). The merits of minimal sample volume, long reagent shelf life and portable instrument made this method optimal for point-of-care applications.

E-008 Endovascular treatment of flow induced anterior spinal artery aneurysm secondary to aortic coarctation

PurposeNeurological complications associated with coarctation of the aorta are generally intracranial. Less familiar, but equally disastrous are the spinal cord complications of this congenital heart lesion. We report on the...

Leeches, Creatine, Xylitol, Spinal Manipulation, Acupuncture

l p r c m w e h b LEECH THERAPY ASSOCIATED WITH SHORT-TERM PAIN REDUCTION IN LATERAL EPICONDYLITIS Level 2 (mid-level) evidence Reference: Clin J Pain 2011;27(5):442 The medicinal use of leeches (Hirudo edicinalis) dates back thousands of years, here it occupied a central role in the ommon practice of bloodletting. When ttached to a patient, leeches pierce the kin with roughly a hundred sharp teeth, nject a mild anesthetic and an anticoaguant (hirudin), and proceed to contentedly uck blood. A large adult leech consumes n average of 5 to 15 mL of blood in a ingle meal. Because of their relatively ainless and tidy habits, leeches were ofen preferred over the lancet for the effiient removal of small quantities of blood. t was not until the late 19th century that loodletting, and along with it the medicnal leech, was finally abandoned as a ainstay of medical practice.

Modificação de técnica de preparo do plasma rico em plaquetas em cães

Platelet-rich plasma (PRP) has many growing factors that improve tissue regeneration. The goal of this paper is to describe an innovative, quick and simple technique for preparing autogenous PRP from a small amount of blood, which allowing use in dogs. After harvesting 8ml of total blood in five dogs, PRP was obtained using an aseptic technique, which rendered a platelet count two to six times more concentrated than the previous serum platelet count, with final absolute values between 800.000 and 1.600.000/mm3. The technique can be developed quickly, without complications using simple equipments, reaching an ideal platelet concentration and keeping plasma sterility.

Separation of young and mature thrombocytes by a novel immuno-selection method

Our earlier studies on the structural and functional properties of zebrafish thrombocytes have shown that they have many similarities to mammalian platelets. We have also shown that zebrafish have both young and mature thrombocytes as do mammalian platelets. In addition, we have distinguished young thrombocytes from mature thrombocytes microscopically using lipophilic DiI-C18, and have shown that young thrombocytes have higher GPIIb receptor levels. However, at present, there is no immunoselection method to separate young thrombocytes from mature thrombocytes in order to study differences among them, such as mRNA expression levels of thrombocyte specific genes. We developed a novel technique employing specific biotinylated anti-Cy3 antibody against the chromophore of DiI-C18 and using streptavidin magnetic beads to separate young thrombocytes from mature thrombocytes. Our technique separates and differentiates young and mature thrombocytes from whole blood. This method is specific and is effective with small amounts of blood.

Evaluation of two devices for point-of-care testing of haemoglobin in neonatal pigs

In veterinary medicine, point-of-care testing (POCT) techniques have become popular, since they provide immediate results and only small amounts of blood are needed. However, their accuracy is controversial. Pigs are often used for research purposes and accurate measurement of haemoglobin (Hb) is important during invasive procedures. The aim of this study was to evaluate two different Hb POCT devices in neonatal pigs. A prospective study with 57 pigs of 3-6 weeks of age, weighing 4.1-6.2 kg (median 5.1 kg) was performed. Fifty-seven blood samples were analysed for Hb using a conductivity-based and a photometrical POCT device and compared with a photometrical reference method. Statistical analysis was performed with Bland-Altman analysis, Spearman correlation and Passing-Bablok regression analysis. Hb values ranged from 32 to 108 g/L (median 80 g/L) using the reference method. The bias of the photometrical method (HemoCue(®)) to the reference method was -1 g/L, with limits of agreement (LOA) of -7 to 6 g/L. The conductivity-based method (i-STAT(®)) had a bias of -15 g/L with LOA from -24 to -6 g/L. There was a significant association between protein values and the bias of i-STAT versus CellDyn (r(2) = 0.27, P < 0.05) but not with the bias of HemoCue versus CellDyn (r(2) = 0.001, P = 0.79). The lower the protein values were, the lower the Hb values were measured by the i-STAT. The conductivity-based measurement of Hb constantly underestimated Hb values, whereas the photometrical method demonstrated a better accuracy and is therefore more reliable for on-site measurement of Hb in pigs.

Accuracy profile validation of a new method for carbon monoxide measurement in the human blood using headspace-gas chromatography–mass spectrometry (HS-GC–MS)

The aim of our study was to provide an innovative headspace-gas chromatography–mass spectrometry (HS-GC-MS) method applicable for the routine determination of blood CO concentration in forensic toxicology laboratories. The main drawback of the GC/MS methods discussed in literature for CO measurement is the absence of a specific CO internal standard necessary for performing quantification. Even if stable isotope of CO is commercially available in the gaseous state, it is essential to develop a safer method to limit the manipulation of gaseous CO and to precisely control the injected amount of CO for spiking and calibration. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in a vial in situ, an internal labeled standard gas (13CO) formed by the reaction of labeled formic acid formic acid (H13COOH) with sulfuric acid. As sulfuric acid can also be employed to liberate the CO reagent from whole blood, the procedure allows for the liberation of CO simultaneously with the generation of 13CO. This method allows for precise measurement of blood CO concentrations from a small amount of blood (10μL). Finally, this method was applied to measure the CO concentration of intoxicated human blood samples from autopsies.

Fluorescent Cell Barcoding as a Tool to Assess the Age-Related Development of Intracellular Cytokine Production in Small Amounts of Blood from Infants

Fluorescent Cell Barcoding (FCB) is a flow cytometric technique which has been used for assessing signaling proteins. This FCB technique has the potential to be applied in other multiparameter analyses. Since data on antigen (Ag)-specific T-cell immune responses, like intracellular cytokine production, are still lacking in infants because limited blood volumes can be obtained for analysis, the FCB technique could be very useful for this purpose. The objectives of this study were to modify the FCB method to be able to measure multiple Ag-specific cytokine reponses in T-cells upon simultaneous stimulation by various antigens and mitogens in small amounts of blood and to investigate the cytokine pattern of T-cell subsets in healthy infants aged six and twelve months. Blood samples, collected from 20 healthy infants aged six and twelve months, were stimulated in vitro with the antigens: phorbol-myristate-acetate (PMA), purified-protein-derivative (PPD), Tetanus-toxoid (TT), Staphylococcal-enterotoxin-B (SEB), and phytohemagglutinin (PHA). Each stimulus was barcoded by labelling with different intensities of fluorescent cell barcoding (FCB) markers. Intracellular production of interleukin-2, interferon-gamma, and tumor necrosis factor-alpha was measured simultaneously in just one blood sample of 600 µl whole blood. Significant age-related differences in cytokine production were shown for PMA, PHA, and TT in CD4+ T-cells, and for PMA, PHA, SEB, and TT in CD8+ T-cells. The intracellular cytokine production by CD4+ and CD8+ T-cells was higher at twelve months compared to six months of age for all antigens, except for PMA, which was lower at the age of twelve months. Based on the consistency in both T-cell subsets, we conclude that the new FCB method is a promising tool to investigate the age-related development of intracellular cytokine production in infants.

Evaluation of the Dried Blood Spot (DBS) Collection Method as a Tool for Detection of HIV Ag/Ab, HBsAg, anti-HBs and anti-HCV in a Malaysian Tertiary Referral Hospital

Dried blood spot (DBS) collection is an appealing alternative to whole blood or plasma sampling, as it has technical and economic advantages over the latter. A prospective cross-sectional study was conducted at a Malaysian tertiary referral hospital from November 2009 to March 2010. One hundred and fifty paired specimens of DBS and plasma were analysed by the standard assays for HIV Ag/Ab, HBsAg, anti-HBS and anti-HCV, separately (total 600 paired specimens). DBS sample titres were then compared to the results of plasma testing, which was used as the gold standard. For the HIV Ag/Ab assay with a cut-off point of 0.35 Relative Light Units (RLUs), the sensitivity and specificity were both 100%. For the HBsAg assay, the sensitivity was 96.5% and the specificity was 97.8%, with a cut-off point of 1.72 RLUs. Sensitivity for the anti-HBs test was 74.2% and the specificity was 86.9%, using a cut-off point of 0.635 RLUs. For the anti-HCV assay, the sensitivity was 97.3% and the specificity was 100%, with a cut-off point of 0.10 RLUs. DBS is an ideal choice to be used as a screening tool for the detection of HIV, Hepatitis B and Hepatitis C virus infections. However, different cut-off values need to be used for the validation of test positivity in DBS samples because the small amount of blood in the DBS specimens leads to lower assay titres.

Commentary: Late Aortic Rupture:The Achilles' Heel of Endovascular Abdominal Aortic Aneurysm Repair

Endovascular aneurysm repair (EVAR) of abdominal aortic aneurysms (AAA) was introduced 20 years ago as a minimally invasive prophylactic procedure to prevent aneurysm rupture. However, rupture after EVAR has been reported and is associated with a high mortality. Rupture after successful EVAR is usually preceded by endograft-related complications, such as type I endoleak and stentgraft migration detected by optimal imaging surveillance. In this issue of JEVT, Venermo et al. attempt to calculate the risk of AAA rupture after EVAR with persistent type Ia endoleak compared to the risk in a cohort of patients with untreated AAA of a similar size. The conventional wisdom is that type I endoleak increases aneurysm rupture risk after EVAR equal to that of a non-operated AAA and therefore needs endovascular reintervention or open conversion. Therefore, the findings that the rupture rate and aneurysm-related death are lower in patients with type I endoleaks compared to the untreated AAA group are interesting and challenge the commonly held notion that type I endoleak following EVAR is evidence of inadequate treatment. This observation suggests that EVAR is beneficial to the patient even if the deployment is not technically successful, which may be of importance in the high-risk patient with multiple comorbidities, a large AAA, and anatomy hostile to stent-graft repair. The authors report that the aneurysmrelated mortality was significantly reduced in the endoleak group compared to the untreated AAA cohort at all time points up to 3 years. The surveillance imaging protocol was good in that the investigators used both duplex ultrasound and contrast-enhanced computed tomography at each follow-up interval with independent classification of endoleak type by both a radiologist and vascular surgeon, although no misclassification rate is given. The length of follow-up is, however, relatively short; ruptures occur at all times during follow-up, with the mean interval from EVAR to rupture recently reported at 4.8 years. The importance of long-term 10year follow-up has been recently highlighted, and the patients in this study require further surveillance before we can draw robust conclusions. As the authors acknowledge, the potential mechanism of protection from rupture despite a persistent type I endoleak is unknown. It may involve prevention or limitation of AAA enlargement by endograft placement. Good stent position, despite an imperfect sealing zone, may allow only a small amount of blood to flow into an already depressurized aneurysm sac, limiting AAA enlargement. The cohort from this study was a ‘‘controlled’’ group of endoleak patients, whereby prompt secondary endovascular intervention was undertaken to try and seal proximal sites that gave rise to type Ia endoleaks. Hence, the

Precision of a point of care testing device for lipid profiling

AbstractPoint‐of‐care testing (POCT) devices can be used to both screen for dyslipidaemia and to monitor lipid levels of patients currently being treated for elevated cholesterol. This study aims to examine the precision of a POCT device when used in a primary care setting. Health screens were offered to all staff members of University College Cork, Ireland. Capillary whole blood samples were taken from two digits of each participant to assess the lipid profile using the POCT device. The relationship between both results was investigated using Pearson product‐moment correlation coefficient, paired sample t‐tests and Kappa measures of agreement. In the second part of the study a volunteer provided ten consecutive capillary whole blood samples. These results were used to calculate the co‐efficient of variation of the device. Data were collected from 55 participants, 25(45%) of whom were male. The mean age of the study population was 44.5 ± 10.1 years. There were statistically significant differences recorded between the results for TC, TG and LDL‐C. Coefficients of variation for TG and LDL‐C were calculated at 7.51 and 7.71%, respectively. There is a degree of variability associated with the precision of POCT device; measurement error is a problem associated with cholesterol testing.Practical applications: Point‐of‐care testing devices are widely used to measure cholesterol levels. They are convenient for patients because only a small amount of blood is required, reducing discomfort. They also have the advantage of providing results quickly, at a location convenient and easily accessible to the patient. It is important that a POCT device being used for the purposes of screening and monitoring is both accurate and precise. This study aimed to examine the precision of a POCT device when used in a primary care setting. The results of this study are consistent with other studies that indicate that measurement error is a problem associated with cholesterol testing. If POCT devices are being used for screening or monitoring purposes then it is necessary to carefully calibrate the device in order to eliminate as much error as possible. Multiple readings should be performed using the same device to identify any potential outlier results.

Ultrasound-assisted translaryngeal block for awake fibreoptic intubation

To the Editor, The difficulty of identifying landmarks can preclude the success of airway blocks and awake tracheal intubation in patients with challenging airways. We describe a case wherein the use of ultrasound made it possible to perform a translaryngeal block successfully by injection of local anesthetic into the tracheal lumen. This technique allowed subsequent awake intubation in a super morbidly obese patient. Informed consent was obtained from the patient prior to submission of this correspondence. The patient was a 38-yr-old female with a body mass index of 57.4 kg m who was scheduled to undergo a Roux-en-Y gastric bypass. Due to a potential difficult airway, the anesthetic plan was to perform an awake fibreoptic intubation. Preoperatively, the patient’s pharynx was sprayed with a combination of 14% benzocaine and 2% tetracaine. In the operating room, the patient was sedated with midazolam 3 mg iv and ketamine 20 mg iv. She was positioned with her neck extended. An attempt was made to identify the cricothyroid membrane by palpating the space between the cricoid and thyroid cartilages. The midline of the membrane was also located by visual inspection and lidocaine was applied subcutaneously. A 20G catheter over a needle attached to a syringe was slowly advanced through the skin while simultaneously attempting to aspirate air. No air could be aspirated, but a small amount of blood (\1 mL) was obtained. At this point, we reassessed the patient’s anatomy and made another attempt (approximately 1.5 cm below the initial attempt) without success. The translaryngeal block was abandoned and fibreoptic intubation was attempted. Even after injection of 4% lidocaine 4 mL through the side port of the fibrescope, the patient still had a prominent cough reflex which precluded the advancement of the fibrescope below the vocal cords. A portable ultrasound machine, Logiq e, (GE Healthcare, Chalfont, St. Giles, UK) was then brought to the operating room, and the patient’s neck was scanned in an attempt to identify landmarks and the midline. A transverse scan was performed at the level of the thyroid and cricoid cartilages (Figure), and the space immediately below the midline of the thyroid cartilage was marked with a pen. The thyroid cartilage had a hypoechoic inverted V-shape appearance, and the cricoid cartilage had a hypoechoic arch-like appearance. A 20G angiocath was inserted through the patient’s skin and air could then be aspirated into the syringe. The catheter of the angiocath was advanced into the larynx and the metal needle was removed. The patient was asked to exhale; 4% lidocaine 4 mL was injected through the catheter at the end of exhalation and the patient coughed profusely. The fibrescope was then advanced through the patient’s mouth, and the vocal cords were easily visualized. The fibrescope was then inserted below the vocal cords without triggering a cough reflex, and a size 7.0-mm endotracheal tube was passed easily through the vocal cords. Correct placement of the endotracheal tube was confirmed with the fibrescope. Examination of the patient’s neck revealed that the first two attempts were made lateral to the midline. In this case, the use of ultrasound made it possible to perform a translaryngeal block in a patient whose landmarks could not be identified correctly using traditional palpation. This case illustrates a potential clinical application for ultrasound assistance in regional anesthesia G. S. De Oliveira Jr, MD (&) P. Fitzgerald, MS M. Kendall, MD Northwestern University, Chicago, USA e-mail: g-jr@northwestern.edu

Easy Diagnosis of Invasive Pneumococcal Disease

To the Editor: Invasive pneumococcal disease (IPD) causes many cases of severe disease and death among children <5 years of age, mostly in developing countries (1,2). Before conjugate vaccines can be introduced in developing countries, information about disease epidemiology is urgently needed. The lack of laboratories equipped to perform pneumococcal serotyping leads to the need to send isolates to reference laboratories. Good sample preservation is necessary to prevent samples from arriving at the laboratory in poor condition. We evaluated the usefulness of multiplex real-time PCR from strains and blood samples kept at room temperature on dried blood spot (DBS) filter paper for detecting and serotyping Streptococcus pneumoniae. DBS screening is a reliable method that requires only a small amount of blood; it is used for the diagnosis of several human diseases (3,4). To validate the technique, we selected 15 pneumococcus clinical isolates representing 15 serotypes (1, 5, 19A, 19F, 14, 3, 7F, 4, 6A, 6B, 8, 9N, 18C, 23A, 23F) obtained during 2009 from patients at Hospital Sant Joan de Deu, in Barcelona. These isolates, used as controls, had been serotyped by quellung reaction at the Instituto de Salud Carlos III, Majadahonda-Madrid, Spain. These strains were cultured overnight at 35°C in 5% carbon dioxide on Columbia agar plates with 5% sheep blood (bioMerieux SA, Marcy l’Etoile, France). A suspension of each strain was adjusted to match a 0.5 McFarland standard (equivalent to 108 colony-forming units (CFU)/mL). Stock solutions of pneumococcus culture for each previously identified serotype were injected into blood previously extracted from 2 healthy volunteers. Serial dilutions of 100,000 CFU/mL to 1,000 CFU/mL (1,000 to 10 CFU equivalents/PCR) were performed. A total of 100 µL of blood was applied to DBS filter paper, and another 100 µL was used for DNA extraction from fresh blood. All DBS samples were air dried for 1 week. The procedure was also performed on negative control blood samples. DNA was extracted from DBS and fresh blood samples by using the NucliSense easyMAG automated extraction platform (bioMerieux, Boxtel, the Netherlands) according to the manufacturer’s instructions. DNA detection of the pneumolysin (ply) gene by real-time PCR was performed according to a published assay (5). In addition, we performed a multiplex real-time PCR for molecular serotype detection of serotypes 1, 3, 5, 4, 6A, 6B, 7FA, 8, 9VANL, 14, 15BC, 18CB, 19A, 19FBC, 23F, 23A and the conserved capsular gene wzg as described by Tarrago et al. (6). DNA extracts were amplified with the Applied Biosystems 7300 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Negative results were defined as those with cycle threshold >40. To evaluate the reliability obtained with this in vivo approach, we performed identification and serotyping of S. pneumoniae in 25 DBS samples from 25 children at Saint John of God Hospital in Mabesseneh-Lunsar, Sierra Leone. This hospital does not perform blood cultures. IPD was confirmed when DNA of a pneumolysin (ply) gene and an additional capsular gene of S. pneumoniae were detected by multiplex real-time PCR of DBS samples. Detection of ply, wzg, and the specific gene for molecular serotype showed that both fresh blood and DBS samples yielded correctly positive results from the 10-fold serial dilutions analyzed (Table). With respect to the 25 (11 female and 14 male) patients from Sierra Leone who had suspected IPD, the median age was 25.71 months (range 15 days to 96 months); all had a diagnosis of fever without apparent source, and 16 also had malaria. Of these 25 children, DBS samples from 15 (60%) yielded a positive result for the ply and wzg genes, so they were considered confirmed episodes of IPD. A serotype included in 13-valent conjugate vaccine was detected in 6 (40%) of 15 positive samples: serotypes 3, 7FA, 19A, 6A, 6B, and 9VNL (1 sample each). In the remaining 9 samples, the results for ply gene and wzg gene were positive, but none of the 24 tested serotypes was detected. Table Sensitivity of real-time PCR for detecting Streptococcus pneumoniae ply or wzg genes or a specific gene for molecular serotype from fresh or dried blood spot samples* This preliminary study enabled us to demonstrate that DBS screening is a reliable and easy method for diagnosing IPD and also for epidemiologic surveillance of the more frequent serotypes. The main limitation of our study is the small number of DBS samples sent from Saint John of God Hospital in Sierra Leone. In conclusion, the DBS technique enables reproducible transport of samples for identification and serotyping of S. pneumoniae by multiplex PCR. The use of DBS on filter paper is an attractive alternative method for storing samples at room temperature and easily transporting them. Additional studies, including evaluation of the relative sensitivity of this method compared to direct culture, are necessary.

Assessing renal function with a rapid, handy, point-of-care whole blood creatinine meter before using contrast materials

The aim of this prospective study was to assess the reliability of a rapid, handy, point-of-care whole blood creatinine meter (PCM) in patients who were scheduled to undergo contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI). Among patients scheduled to undergo contrast-enhanced CT or MRI examinations, 113 patients who did not have creatinine data from the prescribed intervals before the examination day (in principle, 90 days for scheduled outpatients and 7 days for inpatients and urgent patients) were included. The estimated glomerular filtration rate (eGFR) was calculated using the creatinine values measured with the PCM and those from central laboratory measurements (LAB). The two eGFR values were compared statistically with the paired t-test, Pearson's correlation coefficient, and the Bland-Altman analysis. The mean eGFR measured with the PCM was slightly higher than the LAB value (81.2 ± 24.6 vs. 70.2 ± 19.7 ml/min/1.73 m(2), P < 0.0001). They were well correlated (r = 0.74, y = 0.92x + 16.9, P < 0.0001). Bland-Altman plots (mean difference was 11.0 ml/min/1.73 m(2); limits of agreement were -22.4 to +44.4 ml/min/1.73 m(2)) showed a moderate agreement with some degree of dispersion. The PCM can rapidly assess renal function using a small amount of blood almost equally to that of determined in the laboratory, which may help reduce the risk of contrast material-induced complications.

From the lab to the bedside: The present of research, i.e. the future of neonatology

During the last decades, almost every field of neonatology has been the subject of intense investigation. Clinical, epidemiological, and imaging studies, together with experimental research have exponentially increased our knowledge on neonatal physiology and pathology. The most evident effect of the acquisition of this new knowledge has been the great improvement in neonatal care, which has allowed the survival of infants with severe, life threatening diseases, and the prevention of poor long term outcomes (two remarkable examples are theuseof systemichypothermia for thepreventionof hypoxic–ischemic encephalopathy, and the use of less invasive ventilation strategies for the prevention of chronic lung disease, CLD, after extremely premature birth). By translating new knowledge into clinics, in the twentieth century, neonatologists signed the end of the empirical approach to the care of the neonate, and opened the era of a modern neonatology based on clinical and experimental evidence. Much has been done, but much has still to be done. Clinical and epidemiological studies (including instrumental investigations, application of imaging techniques and physiological studies of lung function) are invaluable in our understanding of neonatal development and neonatal diseases. However, a stronger information and a better insight into pathogenic mechanisms of disease can be achieved if clinical and epidemiological data from clinical studies are confirmed by experimental work, or, in turn, if experimental results are confirmed by clinical trials. For many reasons, experimental research in the neonate is difficult. First, the neonate is small: as an example, the entire blood volume is small (approximately 80–100 ml/kg), and can be as small as approximately 40 ml (total) in the extremely pretermneonateweighing 500 g. It is obvious that withdrawal of even small amounts of blood (1–2 ml), could be non-ethical when the clinical conditions of the infant are critical. As a consequence, our possibility to perform research works on plasma or blood cells is someway limited, especially in preterm neonates. An important source of biological samples is the cord blood. However, the cord blood from term infants is often given for donation to blood banks and thus is not available for research, and the cord blood from a very preterm neonate is often difficult to obtain (it is available in smaller amounts, or it is difficult to obtain from the tiny and small umbilical cord of a very pretermneonate born in an emergency setting).