Few Drops Of Blood Research Articles

Ultra-High Performance Liquid Chromatography Method for Bioanalysis of Fampridine Using Dried Blood Spot (DBS) Methodology: Application to Pharmacokinetic Study in Albino Rats.

Fampridine (dalfampridine) is used to improve walking in people who have multiple sclerosis (a disease in which the nerves do not function properly and may cause weakness, numbness, loss of muscle coordination and problems with vision, speech and bladder control). Measurement of fampridine plasma concentrations is not practical at sites lacking the facilities to prepare and process blood samples. A dried blood spot (DBS) sampling method, in which a few drops of blood, drawn by lancet from the finger, are applied onto specially manufactured absorbent filter paper, can be used as an alternative to plasma monitoring and would allow for simplified sample storage and transport. Using blood samples from pharmacokinetic studies, an ultra-high performance liquid chromatography assay method for quantification of fampridine in DBS is developed and validated for specificity, selectivity, accuracy, precision, reproducibility and stability. Method was specific and selective relative to endogenous compounds, with required process efficiency, and no matrix effect. Inaccuracy and precision for intra-day and inter-day analyses were tested at all concentrations. Quantification of fampridine in DBS assay was not affected by blood deposit volume and punch position within spot, and hematocrit level had a limited but acceptable effect on measurement accuracy. Fampridine was stable for at least 2months at room temperature. The correlation between DBS and plasma concentrations with an average blood-to-plasma ratio is determined. DBS sampling is a simple and practical method for monitoring fampridine concentrations. The method is completely validated as per ICH guidelines and extended to the in vivo determination of fampridine in male albino rats.

Love Magic: Spells Using Menstrual Blood in Lithuania

Among menstrual superstitions circulating in Lithuania a belief that it is possible to put a spell on a man using menstrual blood is the least examined. Our article proposes to describe and analyse this superstition. Analysis is based on individual interview data collected in between 2000 and 2020. The number of respondents totals 323. All of them are women. Respondents were asked about the use of menstrual blood for love magic. Only 67 replied that they knew nothing about it. Respondents who learned about blood spells in their youth many decades ago stated that rumours of bewitchment are still circulating even now. The attitude to blood spells is strictly negative. Respondents who thought that the spells were absolutely ineffective regarded them as something stupid and dirty; believers in the power of menstrual blood magic thought that the spells were dangerous, immoral or even equivalent to witchcraft. Adding a few drops of blood to a drink or other liquid is a well-known practice in Lithuania. Less often blood is added to food. The consequences can be tragic: a handsome and bright youth becomes addicted to an old hag; a model husband and father leaves his family. Unfavourable physical effects are also common with spells, for example, triggering serious diseases. Relationships with the seductress are unsuccessful. The removal of a love spell based on menstrual blood is intricate; the techniques of doing so that we recorded are scant, filthy and risky.

Bleeding index and the influence on the value of its various factors

The issue of bleeding is so complex that, despite the considerable literature in this area, it is still poorly developed, and yet not only theoretically, but also purely practically, this issue is of great interest: we often see from everyday surgical practice that with the same size and localization of the wound, one organism bleeds profusely, while the other will have only a few drops of blood.

A low-cost bilirubin measurement tool for neonatal jaundice monitoring at the point-of-care: a comparison of BiliDx with a standard laboratory bilirubinometer and transcutaneous bilirubinometer

BackgroundNeonatal jaundice, defined as an excess of bilirubin in blood at birth or shortly thereafter, has a disproportionately high impact in low-resource settings. Three quarters of deaths from jaundice occur in sub-Saharan Africa and south Asia. Jaundice is easily treated with blue light phototherapy; however, accurate, low-cost bilirubin monitoring is essential to guide effective phototherapy treatment in low-resource settings. To address this need, we developed BiliDx, a low-cost, portable reader that quantifies bilirubin concentration in a few drops of blood applied to a lateral flow card. BiliDx measures absorbance at three wavelengths: blue (bilirubin), amber (haemoglobin interference), and red (background) and can perform measurements in less than 5 mins at the point of care, without the need for a costly centrifuge or extensive training. Here, we compare the accuracy of BiliDx against a standard laboratory bilirubinometer and a transcutaneous bilirubinometer. MethodsThis study took place in two central hospitals in Malawi. We included samples from neonates being treated in neonatal wards and deemed to be at risk for jaundice or who were undergoing phototherapy. Guardians provided written informed consent. We measured bilirubin using three devices: BiliDx, a bilirubinometer (Reichert UNISTAT), and a transcutaneous bilirubinometer (Drager JM-105), with a total of 475 measurements per device. Training (149 measurements) and validation (326 measurements) sets were created by ordering the first measurement per patient from lowest to highest UNISTAT bilirubin; every third measurement was selected for the training set. The training set was used to fit a model that correlated bilirubin concentration to absorbances at three wavelengths. The validation set was used to make an unbiased evaluation of the accuracy of the model. FindingsWe enrolled 456 neonates between August 7 and December 12, 2019; 247 of 326 (75%) of BiliDx measurements in the validation set were within 2 mg/dL of UNISTAT measurements, compared with 44 of 281 (6%) of quantitative transcutaneous measurements. No adverse events occurred. InterpretationBiliDx can provide accurate bilirubin monitoring. BiliDx is more accurate than transcutaneous bilirubinometry, a technology often endorsed for its non-invasive, portable nature. Limitations include that only ten nurses used BiliDx and only central hospitals were included. Testing of the BiliDx device is ongoing; we aim to collect enough measurements >20 mg/dL to assess the accuracy of BiliDx over the intended bilirubin concentration range of 0–35 mg/dL. Future studies will evaluate usability among a larger group of nurses and hospital settings. FundingUSAID Saving Lives at Birth Award No. 7200AA18FA0016.

LSPR based on-chip detection of dengue NS1 antigen in whole blood.

The development of a biosensor for rapid and quantitative detection of the dengue virus continues to remain a challenge. We report a lab-on-chip device that combines membrane-based blood plasma separation and a localized surface plasmon resonance (LSPR) based biosensor for on-chip detection of dengue NS1 antigen from a few drops of blood. The LSPR effect is realized by irradiating UV-NIR light having a spectral peak at 655 nm onto nanostructures fabricated via thermal annealing of a thin metal film. We study the effect of the resulting metal nanostructures on the LSPR performance in terms of sensitivity and limit of detection, by annealing silver films at temperatures ranging from 100 to 500 °C. The effect of annealing temperature on the nanostructure size and uniformity and the resulting optical characteristics are investigated. Further, the binding between non-targeted blood plasma proteins and NS1-antibody-functionalized nanostructures on the LSPR performance is studied by considering different blocking mechanisms. Using a nanostructure annealed at 200 °C and 2X-phosphate buffer saline with 0.05% Tween-20 as the blocking buffer, from 10 μL of whole blood, the device can detect NS1 antigen at a concentration as low as 0.047 μg mL−1 within 30 min. Finally, we demonstrate the detection of NS1 in the blood samples of dengue-infected patients and validate our results with those obtained from the gold-standard ELISA test.

QUALITY OF DRIED BLOOD SPOTS IS AN INTEGRAL COMPONENT OF PROMPT DETECTION OF INBORN ERRORS OF METABOLISM

Introduction. Inborn Errors of Metabolism (IEM) are constituted a group of genetic diseases that are associated with defects in the synthesis or catabolism of complex molecules, impaired intermediary metabolism and energy production/utilization processes. The clinical manifestation of IEM is nonspecific, that looks similar to septicemia, and most often occurs in the neonatal period with life-threatening acute metabolic crises. Expanded Newborn Screening (ENS) – a biochemical study of the blood of all newborns without exception with the purpose to identify molecular markers of these diseases proved to be the most effective instrument of early IEM diagnostics. The quality of the biological samples (dried blood spots, DBS) in great extent determines the timing, accuracy, and reliability of the results of biochemical measurements. Obtaining of equivocal results in the case of analysis of poor quality DBS requires repeated laboratory tests, that delays the diagnostic process and postpones the start of specific treatment, which usually results in irreversible damage of the brain and internal organs of the child.
 The aim of this work is to (i) review the first results of the implementation of Expanded Newborn Screening in Ukraine (pilot part of the Baby Screen Project), and to analyze literature data regarding the negative impact of poor quality DBS on laboratory determination of IEM marker substances contents in the specimens, (ii) to characterize the typical errors in blood sampling and drying of blood spots, and (iii) to provide practical recommendations for the proper performance of these procedures.
 Materials and methods. Own data of retrospective analysis of the questionable ENS results was superimposed with dried blood specimens, that were investigated in the Pharmbiotest ENS Lab to outline most common inaccuracies. Based on the comparison of these data with the relevant publications it was formulated the practical recommendations for improving quality of DBS preparation to ensure the accuracy and reliability of laboratory measurements and speed up IEM diagnostics.
 Results. The quality of biomaterial selection is an important part of obtaining reliable results during expanded newborn screening. Capillary blood is collected in the maternity hospital from 48-72 hours (full-term) and for 7-11 days (in preterm babies) after the birth from the heel of babies. In this case, a few drops of blood are applied to a special test card made of filter paper, which is dried and sent to the laboratory. Blood tests are performed using a highly sensitive and accurate method of chemical analysis - tandem mass spectrometry in the laboratory "Pharmbiotest", located in Ukraine. Taking into analysis the low-quality samples lead to questionable results, which requires repeated DBS sampling and re-examining. This proved to be the most common cause of delaying IEM detection, diagnosis establishment, and initiation of treatment, which can be fatal for a child with severe IEM forms.
 Conclusions. Informing healthcare professionals and parents about the current results of laboratory monitoring of dried blood spots quality, typical errors in blood sampling and following on-site procedures and negative consequences of its improper performance, as well as providing clear practical recommendations of how these procedures should be done is a proven way of improving and speeding up IEM diagnostics.

Abstract PR12: Detection of EV-based signatures in prostate cancer using microflow cytometry and machine learning

Abstract Microflow cytometry (µFCM) has recently emerged as a viable technology to enumerate and quantify individual EVs from complex biologic fluids. This method is a promising alternative to biopsy collection because it is highly sensitive and allows for longitudinal assessment of patients. EVs are highly abundant and analysis by µFCM allows for sophisticated multiparametric analysis of organ-specific, disease-specific, and outcome-specific proteins, genes, or metabolites. Detection of EVs with µFCM requires very little sample yet provides exceptional specificity and sensitivity for low abundance events. By combining µFCM analysis of EVs with advanced machine learning approaches, we have developed a platform technology called ClarityDX to generate EV fingerprints that significantly improve the diagnosis of diseases such as prostate cancer using a few drops of blood. Here I will discuss the development and clinical validation of new blood tests for prostate cancer that utilize the ClarityDX platform. Prostate cancer is the most commonly diagnosed cancer and the third leading cause of cancer deaths in men. Screening has enabled the early detection of many latent prostate cancers, and it is estimated that up to 50% of new prostate cancer diagnoses detect a tumor that was unlikely to surface clinically in the absence of PSA screening. In the majority of cases, the treatment of indolent cancer causes a patient more harm than good. Consequently, there is an urgent need for a noninvasive test to detect clinically significant prostate cancer, or perhaps even more importantly, to detect metastatic disease. ClarityDX Prostate is a µFCM-based test to predict clinically significant prostate cancer prior to a prostate biopsy. A prospective clinical validation of ClarityDX Prostate was performed in a cohort of 377 Albertan men. The overall average AUC for ClarityDX Prostate in this population was 0.83, which was significantly higher than PSA alone. While PSA alone provided only 17% specificity for aggressive prostate cancer at a 95% sensitivity, ClarityDX Prostate was 56% specific for aggressive prostate cancer. We are now conducting a pivotal clinical validation study, in which up to 2,800 men will be enrolled to assess the performance of ClarityDX Prostate in a “real-world” clinical environment. Overall, we have established a robust µFCM EV platform for the development of novel and clinically viable diagnostic tests. Incorporating ClarityDX Prostate into routine prostate cancer screening could reduce the number of unnecessary biopsies by up to 40%. This abstract is also being presented as Poster B57. Citation Format: John D. Lewis, Robert J. Paproski, Desmond B. Pink, Catalina Vasquez, Eric Hyndman, Adrian Fairey, APCaRI. Detection of EV-based signatures in prostate cancer using microflow cytometry and machine learning [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR12.

Amantadine hydrochloride monitoring by dried plasma spot technique: High-performance liquid chromatography-tandem mass spectrometry based clinical assay.

Amantadine plasma concentrations correlate well with desired therapeutic effects and adverse outcomes; information on amantadine exposure could be useful when multiple amantadine clearance pathways are impaired or non-compliance is suspected. Micro-sampling strategies, like dried plasma spot, would be particularly useful because ambulatory patients that do not attend a clinic can easily sample a few drops of blood by themselves at the required time of the dosing interval. We developed and validated a dried-plasma-spot-based high performance liquid chromatography-tandem mass spectrometry assay to quantify amantadine. This assay met relevant validation requirements within a hematocrit range of 20-50% and was linear from 100 to 2000ng/mL. Amantadine was stable in dried plasma spots for up to 21 days at room temperature, regardless of whether the dried plasma spot was protected from light or not. The correlation between paired dried and wet plasma concentrations was assessed in 52 patients. Deming regression coefficients between wet plasma and simultaneously pipetted dried plasma spots were used to predict plasma concentrations. Bland-Altman plots revealed a strong agreement between dried and wet plasma concentrations, supporting the clinical usefulness of dried plasma spots for amantadine monitoring with a self-sampling strategy at a convenient time and place for the patient.

Galactosemia.

Galactosemia.

Carbon nanofiber-based multiplexed immunosensor for the detection of survival motor neuron 1, cystic fibrosis transmembrane conductance regulator and Duchenne Muscular Dystrophy proteins

Simultaneous and point-of-care detection of multiple protein biomarkers has significant impact on patient care. Spinal Muscular Atrophy (SMA), Cystic Fibrosis (CF) and Duchenne Muscular Dystrophy (DMD) are well known progressive hereditary disorders associated with increased morbidity as well as mortality. Therefore, rapid detection of biomarkers specific for these three disorders in newborns offers new opportunities for early diagnosis, delaying symptoms and effective treatment. Here, we report the development of a disposable carbon nanofiber (CNF)-based electrochemical immunosensor for simultaneous detection of survival motor neuron 1 (SMN1), cystic fibrosis transmembrane conductance regulator (CFTR) and DMD proteins. The CNF-modified array electrodes were first functionalized by electroreduction of carboxyphenyl diazonium salt. Then, the immunosensor was fabricated by the covalent immobilization of the three antibodies on the working electrodes of the array sensor via carbodiimide (EDC/NHS) chemistry. Simultaneous detection of CFTR, DMD and SMN1 was achieved with high sensitivity and detection limits of 0.9 pg/ml, 0.7 pg/ml and 0.74 pg/ml, respectively. The multiplexed immunosensor has also shown strong selectivity against non-specific proteins. Moreover, high recovery percentage was obtained when the immunosensor was applied in spiked whole blood samples. This voltammetric immunosensor offers cost effective, easy to use, rapid and high throughput potential screening method for these three hereditary disorders using only few drops of blood.

Theranos Experience Exposes Weaknesses in FDA Regulatory Discretion.

Theranos Experience Exposes Weaknesses in FDA Regulatory Discretion.

Improving Reproducibility of Lab-on-a-Chip Sensor with Bismuth Working Electrode for Determining Zn in Serum by Anodic Stripping Voltammetry

This work reports on the continuing development of a lab-on-a-chip electrochemical sensor for determination of zinc in blood serum using square wave anodic stripping voltammetry. The microscale sensor consists of a three electrode system, including an environmentally friendly bismuth working electrode, an integrated silver/silver chloride reference electrode, and a gold auxiliary electrode. The sensor demonstrates a linear response in 0.1 M acetate buffer at pH 6 for zinc concentrations in the 1–30 μM range. By optimizing bismuth film deposition and better control of the fabrication process, repeatability of the sensor was improved, reducing variability from 42% to <2%. Through optimization of electrolyte and stripping voltammetry parameters, limit of detection was greatly improved to 60 nM. The optimized sensor was also able to measure zinc in the extracted blood serum. Ultimately, with integrated sample preparation, the sensor will permit rapid (min) measurements of zinc from a sub-mL sample (a few drops of blood) for clinical applications.

Piezoelectric rotational mixer based on a first bending vibration mode

We propose a miniature piezoelectric mixer that can rotate a liquid inside a hole of several millimeters diameter. The mixer has dimensions 14 × 14 × 10 mm with a through-hole (7 mm), and piezoelectric elements bonded to its four identical sides. When the first bending vibration mode of the mixer is excited by ac voltages, rotational flow of liquid is generated by the rotation of an acoustic field (acoustic streaming) in the through-hole. This technology is useful in automating mixing processes such as the mixing by hand of a few drops of blood in medical testing. In this paper, we verify the driving principle experimentally and examine the characteristics of a prototype mixer, including the induced flow velocity, under changes of the amplitude and frequency of the applied voltages. A plastic test tube, used to prevent contamination, is inserted to the through-hole, and the liquid inside the tube is mixed by rotation of the acoustic field.

153 DETECTING PRE-OVULATORY LUTEINIZING HORMONE PEAKS IN ORDER TO OPTIMIZE THE RATIO OF VIABLE EMBRYOS USING PREDI′BOV®, A NEW ON-FARM OVULATION TEST

The LH peak in cattle is the most precise event for predicting ovulation beginning 24 h later, and thus, AI time. Previous studies demonstrated that embryo production was improved when AI was conducted 12 h before ovulation; that is, 12 h after LH peak. This study aimed to evaluate the benefit of LH peak monitoring with Predi′Bov® (ReproPharm®, Nouzilly, France) following superstimulation in order to optimize numbers of viable embryos (VE). Predi′Bov® is a rapid (40 min) and easy to use on-farm test allowing LH peak detection from a few drops of blood. The test was also used to estimate the variability in the time of the LH peak and onset of oestrus. This study was conducted by the embryo transfer teams of 3 French cooperatives, in collaboration with UNCEIA. Forty heifers in stations (Creavia, Midatest) and 23 cows on farms (GEN′Iatest) were superstimulated by 8 injections IM of Stimufol® or Pluset® (FSH1 to FSH8) over 4 days in 2011–2012. Donor station heifers were treated twice in a Latin square design with a reference protocol where AI was conducted 12 and 24 h after onset of oestrus, and in an experimental protocol where AI was conducted 12 and 24 h after a positive Predi′Bov® test. Semen of different sires was used for both protocols. The Predi′Bov® test was carried out on 3 blood samples (BS1,2,3) collected every 6 h beginning at FSH7 in stations and FSH8 on farms to detect the earliest LH peaks. To determine late LH peaks, Predi′Bov® test was carried out on BS4 collected 24 h after FSH8. Univariate statistical analysis was performed to look at the relationship between qualitative (chi-square) and quantitative (t-test) variables. The difference was considered significant when P &lt; 0.05. The Predi′Bov® test showed that 37.5% (15/40) of LH peaks occurred during the last day of FSH treatment (BS1 or BS2) in stations and 26.1% (6/23) at BS1 on farms. At Creavia station (n = 24), the LH peak was detected anytime from 24 h before to 9 h after the onset of oestrus. In stations, the VE percentage did not differ whether AI was done following oestrus or LH peak detection (63.1% and 61.8% in reference and experimental protocols, respectively). In stations, the VE percentage from 9 females with an LH peak detected at FSH7 (BS1; 41%) in the reference protocol did not differ from the experimental protocol (50%). On farms, the VE percentage was numerically higher but not significant in the experimental protocol (65.4%, n = 16) compared to the reference protocol (47.2%, n = 7). Further investigations are needed, taking into account the effect of collection rank, sire, and female effects, to confirm the trends shown by these results. In conclusion, Predi′Bov® can be used as easily on farms as in stations. Its use allows the detection of animals that have early or late LH peaks, which in turn provides the opportunity of carrying out AI at the optimal time for such females.

Maintaining Trust in Newborn Screening

AbstractNewborn screening consists of taking a few drops of blood from a baby's heel in the first week of life and testing it for a list of disorders. In the United States and most countries in Europe, newborn screening programs began in the 1960s and 1970s with screening for phenylketonuria (PKU), a rare metabolic disease that causes severe and irreversible mental retardation unless treated before problems arise. As knowledge about rare diseases expanded and new screening technologies were introduced—such as the tandem mass spectrometer and high‐performance liquid chromatography—the same blood sample could be used to test for a whole list of disorders.In general, screening programs in most countries have tended to expand, but in different countries they have expanded in different ways. Regulation also varies. In some states, screening is mandatory, whereas in others—Wyoming and Maryland—parents are asked for their informed consent. Germany and France have adopted an explicit informed consent procedure, whereas other European countries have a more informal “opt‐out” procedure that does not require signing an informed consent form.Whether newborn screening requires informed consent is an ongoing issue in bioethics. In this article, we will focus on the tension between informed consent and the problem of compliance in newborn screening. Asking for informed consent—allowing parents to opt out—is often thought to pose a threat to compliance. Building on the work of Onora O'Neill on informed consent and trust, however, as well as on work she coauthored with Neil Manson, we will argue that informed consent procedures may actually help maintain trust in newborn screening and may therefore support compliance.

The Michigan BioTrust for Health: Using Dried Bloodspots for Research to Benefit the Community While Respecting the Individual

The Michigan Department of Community Health (MDCH) stores almost 4 million dried blood spot specimens (DBS) in the Michigan Neonatal Biobank. DBS are collected from newborns under a mandatory public health program to screen for serious conditions. At 24 to 36 hours of age, a few drops of blood are taken from the baby’s heel and placed on a filter paper card. The card is sent to the state public health laboratory for testing. After testing, MDCH retains the spots indefinitely for the personal use of the patient and also, pursuant to a 2000 law, for possible research.

Out of a Cell into This Darkened Space

Out of a Cell into This Darkened Space

Some recollections about kuru in a patient at Rabaul in 1978, and subsequent experiences with prion diseases

I first heard about kuru when I was undertaking the six-month full-time Diploma in Tropical Medicine and Hygiene (DTM&H) course at Edinburgh University in 1970–1971. The Head of the Department of Infectious and Tropical Diseases, the eminent Dr Frederick J. Wright, had visited Papua New Guinea and

Mass spectrometry in newborn and metabolic screening: historical perspective and future directions

The growth of mass spectrometry (MS) in clinical chemistry has primarily occurred in two areas: the traditional clinical chemistry areas of toxicology and therapeutic drug monitoring and more recent, human genetics and metabolism, specifically inherited disorders of intermediary metabolism and newborn screening. Capillary gas chromatography and electrospray tandem MS are the two most common applications used to detect metabolic disease in screening, diagnostics and disease monitoring of treated patients. A few drops of blood from several million newborn infants are screened annually throughout the world making this the largest application of MS in medicine. Understanding the technique, how it grew from a few dozen samples per week in the early 1990s to increasing daily volume today will provide important information for new tests that either expand newborn screening or screening in other areas of metabolism and endocrinology. There are numerous challenges to the further expansion of MS in clinical chemistry but also many new opportunities in closely related applications. The model of newborn screening and MS in medicine may be useful in developing other applications that go beyond newborns and inherited metabolic disease. As MS continues to expand in clinical chemistry, it is clear that two features will drive its success. These features are excellent selectivity and multiple analyte or profile analysis; features recognized in the 1950s and remain true today.

Ghati‐Bangal on the Maidan: subregionalism, club rivalry and fan culture in Indian football

Indian football’s premier club rivalry between Mohun Bagan and East Bengal mirrors the oppositional identities, social differences and emotional commonalities inherent in Indian society and culture. This paper seeks to suggest that while the roots of the origin and consolidation of this football rivalry lie in social and sub‐regional differences of contemporary Indian society, its playing out in the last 25 years can only be meaningfully explained in terms of club loyalty, fan culture and commercial transformation. It was an unimportant match between East Bengal and a local team in the late 1970s. The match was just about to start. A small crowd was chatting near the main entrance of the ground. Suddenly a middle‐aged man with messy hair, red eyes and a worn out look appeared at the gate. While entering the ground he anxiously asked the gatekeeper whether the match had begun. The gateman replied in the negative. A few other members who knew the man came to him and asked why he was so late in arriving on that day. The man hesitantly replied: ‘My younger son passed away in the morning. I am coming straight from the crematorium.’1 In a League match in the late 1970s, Mohun Bagan won by a big margin. After the match the jubilant crowd rushed towards Esplanade to board buses and trams on the way home. A few bare‐bodied young lads were standing on the rod of the tram’s wheel, hanging on to the windows with one hand. They were waiving their shirts like Mohun Bagan flags. As the tram suddenly stopped at a road signal, one of the lads got thrown onto the street. His feet appeared to be run over by the wheels of the tram. However, as the tram started to move, the lad ran and jumped onto the rod to catch the tram. After the tram left the place, a severed toe was found amidst a few drops of blood while a thin red line stretched along the tramline. The pain got lost in the euphoria of victory.2