The document describes the development of an enzyme assay to differentiate between two strains of Leuser Virus in Southeast Asia. The assay targets enzyme X levels, which are elevated in serum of those infected with the fatal second strain. The assay was optimized for pH, temperature, and incubation time. It was then used to test samples from known positive patients to determine a optimal cutoff level. This level gave 100% sensitivity and 90% specificity. Further samples correctly diagnosed patients, differentiating strains. Compared to the standard ELISA test, the enzyme assay allows differentiation of strains for improved patient care and treatment.
The document describes investigations into developing a diagnostic test for the Leuser virus causing 'yellow-eye' disease. Two strains have emerged - a mild strain and lethal mutant strain. Currently, diagnosis relies on an ELISA blood test which is costly and cannot differentiate strains. The study developed a proof of principle assay shown to be accurate, specific, sensitive and reliable for diagnosing the virus. Further investigations found the virus causes liver damage leading to jaundice, erythrocyte damage from hemolysis, and evidence of kidney damage/infection. While the metabolite P was not a useful diagnostic marker, the optimized assay shows potential as a lower cost diagnostic to replace the current ELISA and help differentiate between virus strains.
The document describes developing an alternative diagnostic test for detecting the severe strain of Leuser virus based on measuring levels of enzyme X in serum. The researchers optimized an enzyme X assay by testing different pH levels, temperatures, time intervals, and wavelengths. They determined an optimal cutoff level using samples from patient Set A and then applied this to classify unknown samples from patient Set B as either mild or severe strain. The enzyme X assay demonstrated 100% sensitivity and 70% specificity. While promising, the researchers noted limitations like low quality control values and sample size that could be addressed in future studies.
This document discusses various laboratory diagnostic techniques for parasitic infections. It begins by noting that light microscopy remains the standard for diagnosis of most parasites, but nucleic acid tests are becoming more popular. Immunological diagnosis using techniques like ELISA and rapid diagnostic tests (RDTs) have advantages like speed and ease of use. The document then examines specific techniques for diagnosing various parasites like malaria, cryptosporidiosis, amoebiasis, trichomoniasis, lymphatic filariasis, and leishmaniasis. It discusses advantages and limitations of different antigen and antibody detection methods.
Internal quality control in blood bank testingSanjeew Yadav
Internal quality control is essential for blood bank testing to continuously monitor laboratory work and results. It involves running both quantitative and qualitative controls to check for issues like drift, dispersion, or shifts that could indicate problems. Quantitative tests have controls run to establish mean, standard deviation, and target ranges using statistical analysis. Qualitative rapid card tests have positive and negative controls run daily. Equipment and reagents also have defined internal quality checks run daily or weekly. Control values are monitored using Levy-Jennings charts and Westgard rules to ensure precision and accuracy and identify any issues requiring troubleshooting or corrective action.
Haemoglobin quality control by maintaining levey jennings chartDr Rashmi Sood
Haemoglobin quality control is important for blood donor selection and ensuring donor safety. The document discusses maintaining Levey Jennings charts to monitor internal quality control of haemoglobin measurements using a Hemocue analyzer. Westgard rules are applied to the charts to detect random errors like those caused by new staff and systematic errors indicating issues like reagent problems. Maintaining quality control through daily, weekly and monthly checks helps assure accurate haemoglobin results.
this is just a review of Quality Control Scheme integrated by Dr Westgard and his son, as here in ordinary clinic lab that QA Scheme is followed, and the LH 500 and our ACT5 Diff Coulter Counter, we are able to get the Yearly Sigma Scale performance for each analytes, our TAE for each analytes, I compute based on Rico's accepted biological variation as I deemed it is much pertinent, thus the EQAS in the peer group, cumulative bias average for 6 months and the IQAS's LJ Chart has the CV%. The two instruments are well maintained , with carryover check for each modes, Accuracy / Precision Check By the Engineer, the Calibration, is always Passed the required threshold for each parameter.
This powerpoint presentation is graciously dedicated to the father and son, the WESTGARDS...we owe you this quality assurance for each instruments we have, either in Biochem or Hematology, we salute you and more power, God Bless. From Sis Rina, our simple JPC lab here in the Gulf--thanks so much!
COMPARISON OF DIAGNOSTIC PERFORMANCES OF THREE COMMERCIAL ELISA KITS FOR DETE...EuFMD
This study compared the diagnostic performance of three commercial ELISA kits for detecting antibodies against foot-and-mouth disease virus type O (FMDV-O) in cattle and pigs in South Korea. Sera from vaccinated and infected animals were tested using the three ELISA kits and results were compared to virus neutralization tests (VNT). The domestic ELISA kits generally showed higher sensitivity than the international kit, though the international kit had higher specificity. All three kits showed moderate to substantial agreement with VNT results. This evaluation of diagnostic performance can help ensure reliable serological surveillance for FMDV-O in South Korea.
This document provides information and guidelines on blood specimen collection through venipuncture. It discusses identifying patients, preparing the collection site and equipment, performing the venipuncture using proper technique, and handling and processing the specimens. Specific topics covered include selecting appropriate vein sites, using tourniquets and antiseptics, avoiding complications, and processing and storing specimens according to their test requirements. The goal is to provide high quality specimens by following standard procedures.
The document describes investigations into developing a diagnostic test for the Leuser virus causing 'yellow-eye' disease. Two strains have emerged - a mild strain and lethal mutant strain. Currently, diagnosis relies on an ELISA blood test which is costly and cannot differentiate strains. The study developed a proof of principle assay shown to be accurate, specific, sensitive and reliable for diagnosing the virus. Further investigations found the virus causes liver damage leading to jaundice, erythrocyte damage from hemolysis, and evidence of kidney damage/infection. While the metabolite P was not a useful diagnostic marker, the optimized assay shows potential as a lower cost diagnostic to replace the current ELISA and help differentiate between virus strains.
The document describes developing an alternative diagnostic test for detecting the severe strain of Leuser virus based on measuring levels of enzyme X in serum. The researchers optimized an enzyme X assay by testing different pH levels, temperatures, time intervals, and wavelengths. They determined an optimal cutoff level using samples from patient Set A and then applied this to classify unknown samples from patient Set B as either mild or severe strain. The enzyme X assay demonstrated 100% sensitivity and 70% specificity. While promising, the researchers noted limitations like low quality control values and sample size that could be addressed in future studies.
This document discusses various laboratory diagnostic techniques for parasitic infections. It begins by noting that light microscopy remains the standard for diagnosis of most parasites, but nucleic acid tests are becoming more popular. Immunological diagnosis using techniques like ELISA and rapid diagnostic tests (RDTs) have advantages like speed and ease of use. The document then examines specific techniques for diagnosing various parasites like malaria, cryptosporidiosis, amoebiasis, trichomoniasis, lymphatic filariasis, and leishmaniasis. It discusses advantages and limitations of different antigen and antibody detection methods.
Internal quality control in blood bank testingSanjeew Yadav
Internal quality control is essential for blood bank testing to continuously monitor laboratory work and results. It involves running both quantitative and qualitative controls to check for issues like drift, dispersion, or shifts that could indicate problems. Quantitative tests have controls run to establish mean, standard deviation, and target ranges using statistical analysis. Qualitative rapid card tests have positive and negative controls run daily. Equipment and reagents also have defined internal quality checks run daily or weekly. Control values are monitored using Levy-Jennings charts and Westgard rules to ensure precision and accuracy and identify any issues requiring troubleshooting or corrective action.
Haemoglobin quality control by maintaining levey jennings chartDr Rashmi Sood
Haemoglobin quality control is important for blood donor selection and ensuring donor safety. The document discusses maintaining Levey Jennings charts to monitor internal quality control of haemoglobin measurements using a Hemocue analyzer. Westgard rules are applied to the charts to detect random errors like those caused by new staff and systematic errors indicating issues like reagent problems. Maintaining quality control through daily, weekly and monthly checks helps assure accurate haemoglobin results.
this is just a review of Quality Control Scheme integrated by Dr Westgard and his son, as here in ordinary clinic lab that QA Scheme is followed, and the LH 500 and our ACT5 Diff Coulter Counter, we are able to get the Yearly Sigma Scale performance for each analytes, our TAE for each analytes, I compute based on Rico's accepted biological variation as I deemed it is much pertinent, thus the EQAS in the peer group, cumulative bias average for 6 months and the IQAS's LJ Chart has the CV%. The two instruments are well maintained , with carryover check for each modes, Accuracy / Precision Check By the Engineer, the Calibration, is always Passed the required threshold for each parameter.
This powerpoint presentation is graciously dedicated to the father and son, the WESTGARDS...we owe you this quality assurance for each instruments we have, either in Biochem or Hematology, we salute you and more power, God Bless. From Sis Rina, our simple JPC lab here in the Gulf--thanks so much!
COMPARISON OF DIAGNOSTIC PERFORMANCES OF THREE COMMERCIAL ELISA KITS FOR DETE...EuFMD
This study compared the diagnostic performance of three commercial ELISA kits for detecting antibodies against foot-and-mouth disease virus type O (FMDV-O) in cattle and pigs in South Korea. Sera from vaccinated and infected animals were tested using the three ELISA kits and results were compared to virus neutralization tests (VNT). The domestic ELISA kits generally showed higher sensitivity than the international kit, though the international kit had higher specificity. All three kits showed moderate to substantial agreement with VNT results. This evaluation of diagnostic performance can help ensure reliable serological surveillance for FMDV-O in South Korea.
This document provides information and guidelines on blood specimen collection through venipuncture. It discusses identifying patients, preparing the collection site and equipment, performing the venipuncture using proper technique, and handling and processing the specimens. Specific topics covered include selecting appropriate vein sites, using tourniquets and antiseptics, avoiding complications, and processing and storing specimens according to their test requirements. The goal is to provide high quality specimens by following standard procedures.
Performance Indicators in Blood Transfusion (Dr. Nashwa Elsayed)Nashwa Elsayed
This document discusses key performance indicators (KPIs) for blood transfusion services. It begins by explaining that KPIs are an important tool for quality management in complex processes like blood transfusion that involve donor recruitment, testing, production of blood products, and clinical use. The document then covers types of KPIs, how to select, define objectives and limits for KPIs, and examples of KPIs used in transfusion medicine like percentage of volunteer donors, donor satisfaction, adverse donor reactions, and rejected blood unit rates.
Quality assurance in relation to medical laboratory accreditationDr. T.A. Varkey
Dr. TA Varkey PhD, Managing Director, Medilab Speciality Laboratories, Kochi explains the need of Quality Control in Clinical Laboratories and how Quality assurance can be made on various procedures done.
The document discusses the conceptual design and experimental setup of a Visible Light Communication system called VIDAS for transmitting traffic information to vehicles. VIDAS uses LED traffic lights to transmit data to onboard vehicle receivers via visible light modulation. Key components discussed include the multiple LED emitter source, PIN photodiode detector, front-end amplifier, direct sequence spread spectrum modulation, and considerations for noise and signal variation over distance. Experimental results showed VIDAS enabled reception of traffic information from 100m away and adaptation to changing signal strength as vehicles approached intersections.
VMA (vanillylmandelic acid) is a metabolite of catecholamines produced in the liver. It is excreted in urine and can be measured to help diagnose catecholamine-producing tumors like pheochromocytoma. There are several methods to measure urinary VMA levels, including spectrophotometry, chromatography, and ELISA. For accurate results, patients are asked to avoid certain foods and medications prior to a 24-hour urine collection. Normal urinary VMA levels are 1.7-7.5 mg/24 hours. Abnormal levels can indicate neuroblastoma or other catecholamine-producing tumors.
Blood screening, quarantine and releaseRafiq Ahmad
The document discusses procedures for screening donated blood for infectious diseases at a regional laboratory and blood bank. It provides details on:
1. The viruses, bacteria, parasites, and prions that are screened for, including HIV, HCV, HBV, HTLV, syphilis, malaria, and vCJD.
2. The screening markers and recommended tests used to detect these infectious agents, such as ELISA, chemiluminescence, and nucleic acid amplification technologies.
3. Protocols for quarantining and properly disposing of reactive blood units based on screening results, as well as archiving donor samples and blood components for further testing and research.
This document provides instructions and results for several microbiology experiments including gram staining common bacteria, observing bacterial cell morphology under microscopy, performing different staining techniques to identify structures like capsules and endospores, assessing bacterial growth under different temperatures and oxygen conditions, and techniques for culturing both aerobic and anaerobic bacteria. The experiments aim to classify and identify different bacteria based on their cellular and colonial morphology as well as their growth requirements.
Quality Assurance of Laboratory Test Results based on ISO/IEC 17025PECB
The document discusses quality assurance of laboratory test results based on ISO/IEC 17025. It discusses the importance of analytical measurement data and ensuring results are accurate and precise. It describes internal quality control procedures like spikes, blanks, and replicates that analysts use to ensure results are correct. It also discusses external quality assurance like participating in inter-laboratory comparisons and proficiency testing schemes. The document emphasizes that laboratories must have quality control procedures to monitor validity of tests and ensure trends in results are detectable.
Please Subscribe to this Channel for more solutions and lectures
http://www.youtube.com/onlineteaching
Chapter 9: Inferences from Two Samples
9.2: Two Means, Independent Samples
This document provides information about transfusion transmissible infection (TTI) testing performed at a laboratory. It describes 5 tests conducted - for HIV, HBV, HCV, syphilis, and malaria. The methods used are ELISA, MEIA, rapid tests, TPHA for syphilis, and slide testing for malaria. Detailed procedures are provided for each test, including sample preparation, controls, interpretation of results, and any special equipment used like the Miniswift or AXSym machines for automated ELISA and MEIA.
This document discusses various special stains used in pathology to identify structures in tissue samples. It provides details on stains for lipids, carbohydrates, iron, myeloperoxidase, alkaline phosphatase, reticulin fibers, and other components. The stains discussed include Sudan Black B, Periodic Acid Schiff, Perl's Iron stain, myeloperoxidase, and reticulin stain. The objectives of special staining are to provide a definitive diagnosis and aid in differential diagnoses of hematological conditions.
IMPLEMENTATION OF QUALITY CONTROL PERFORMANCE CRITERIA AND APPROVED GUIDELINE...Moustafa Rezk
IMPLEMENTATION OF QUALITY CONTROL PERFORMANCE CRITERIA AND APPROVED GUIDELINES FOR UPGRADING OF CLINICAL CHEMISTRY LABORATORY PROCEDURES IN ALEXANDRIA UNIVERSITY HOSPITALS
This document provides information on performing and interpreting a gram stain of sputum samples. It describes the principles behind gram staining, outlines the staining procedure and sources of errors. It also details how to evaluate sputum quality, quantify and identify bacteria, fungi and other cells seen on gram stain. Quality control measures are highlighted to help ensure accurate results.
The document discusses Chi-Square tests, which are used when assumptions of normality are violated. It provides requirements for Chi-Square tests, including that variables must be independent and samples sufficiently large. The key steps are outlined: determine appropriate test, establish significance level, formulate hypotheses, calculate test statistic using frequencies, determine degrees of freedom, and compare to critical value. An example compares party membership to opinions on gun control to demonstrate a Chi-Square test of independence.
ISO 15189 is an international standard that specifies the general requirements for the competence of medical laboratories. It is based on ISO 17025 for testing and calibration laboratories and ISO 9001 for quality management systems. ISO 15189 has both management and technical requirements that medical laboratories must meet in order to be accredited. The standard is designed to ensure that laboratories consistently deliver accurate, reliable and timely medical testing services.
The document describes developing an alternative diagnostic test for detecting the severe strain of Leuser virus based on measuring levels of enzyme X in serum. The researchers optimized an enzyme assay to measure enzyme X levels, determining the best pH, temperature, measurement time and wavelength. They established a cutoff level using samples from patient set A and then classified unknown samples from patient set B as mild or severe strain based on this cutoff. The assay demonstrated 100% sensitivity and 70% specificity. The enzyme X assay shows potential as a rapid, specific and cheaper alternative diagnostic test for the severe strain of Leuser virus.
Validation of Opsonophagocytic assay for pnemococcal vaccinekishor kulkarni
This document describes the validation of an opsonophagocytic killing assay for Streptococcus pneumoniae. The objectives of the validation study were to assess the assay's precision, linearity, accuracy, robustness, and specificity according to ICH guidelines. The results showed that the assay had 100% precision when testing multiple samples, good linearity with correlation coefficients between 0.948-1.000, 100% accuracy when compared to a reference standard, was robust to changes in effector-to-target cell ratios up to 100:1, and demonstrated specificity by inhibiting ≥80% of opsonophagocytic activity with homologous polysaccharides and ≤20% inhibition with heterologous polysaccharides. The conclusion is
This document discusses different types of ELISA results - quantitative, qualitative, and semi-quantitative. It provides details on generating a standard curve for quantitative ELISAs, including examples of pipetting schemes and plotting absorbance values versus analyte concentration. For qualitative ELISAs, it describes comparing sample absorbance values to a cut-off calibrator. The document also gives an example of calculating a cut-off value and criteria for interpreting qualitative ELISA results.
The job market is difficult, but you have managed to obtain a temporary job in a medical research team. You are pleasantly surprised to be told that you were appointed because of your expertise in statistics. The team is investigating the effects of poisonous metals on human health. They will be obtaining data from various sources, but in the meantime they have given you some old data for you to learn about the kinds of analysis required.
The main purpose of this research project was to develop a quantitative serological diagnostic enzyme assay to detect and differentiate between the two strains of Yellow Eye Disease. The research optimized an enzyme assay that achieved a 97.5% diagnostic success rate compared to the current ELISA method. Investigation into the pathogenesis through urinalysis, haemolysis assays, and H&E staining indicated hepatic malfunction and haemolysis in diseased patients. The results provide a foundation to improve both treatment and diagnosis of the more severe strain two of the virus.
Performance Indicators in Blood Transfusion (Dr. Nashwa Elsayed)Nashwa Elsayed
This document discusses key performance indicators (KPIs) for blood transfusion services. It begins by explaining that KPIs are an important tool for quality management in complex processes like blood transfusion that involve donor recruitment, testing, production of blood products, and clinical use. The document then covers types of KPIs, how to select, define objectives and limits for KPIs, and examples of KPIs used in transfusion medicine like percentage of volunteer donors, donor satisfaction, adverse donor reactions, and rejected blood unit rates.
Quality assurance in relation to medical laboratory accreditationDr. T.A. Varkey
Dr. TA Varkey PhD, Managing Director, Medilab Speciality Laboratories, Kochi explains the need of Quality Control in Clinical Laboratories and how Quality assurance can be made on various procedures done.
The document discusses the conceptual design and experimental setup of a Visible Light Communication system called VIDAS for transmitting traffic information to vehicles. VIDAS uses LED traffic lights to transmit data to onboard vehicle receivers via visible light modulation. Key components discussed include the multiple LED emitter source, PIN photodiode detector, front-end amplifier, direct sequence spread spectrum modulation, and considerations for noise and signal variation over distance. Experimental results showed VIDAS enabled reception of traffic information from 100m away and adaptation to changing signal strength as vehicles approached intersections.
VMA (vanillylmandelic acid) is a metabolite of catecholamines produced in the liver. It is excreted in urine and can be measured to help diagnose catecholamine-producing tumors like pheochromocytoma. There are several methods to measure urinary VMA levels, including spectrophotometry, chromatography, and ELISA. For accurate results, patients are asked to avoid certain foods and medications prior to a 24-hour urine collection. Normal urinary VMA levels are 1.7-7.5 mg/24 hours. Abnormal levels can indicate neuroblastoma or other catecholamine-producing tumors.
Blood screening, quarantine and releaseRafiq Ahmad
The document discusses procedures for screening donated blood for infectious diseases at a regional laboratory and blood bank. It provides details on:
1. The viruses, bacteria, parasites, and prions that are screened for, including HIV, HCV, HBV, HTLV, syphilis, malaria, and vCJD.
2. The screening markers and recommended tests used to detect these infectious agents, such as ELISA, chemiluminescence, and nucleic acid amplification technologies.
3. Protocols for quarantining and properly disposing of reactive blood units based on screening results, as well as archiving donor samples and blood components for further testing and research.
This document provides instructions and results for several microbiology experiments including gram staining common bacteria, observing bacterial cell morphology under microscopy, performing different staining techniques to identify structures like capsules and endospores, assessing bacterial growth under different temperatures and oxygen conditions, and techniques for culturing both aerobic and anaerobic bacteria. The experiments aim to classify and identify different bacteria based on their cellular and colonial morphology as well as their growth requirements.
Quality Assurance of Laboratory Test Results based on ISO/IEC 17025PECB
The document discusses quality assurance of laboratory test results based on ISO/IEC 17025. It discusses the importance of analytical measurement data and ensuring results are accurate and precise. It describes internal quality control procedures like spikes, blanks, and replicates that analysts use to ensure results are correct. It also discusses external quality assurance like participating in inter-laboratory comparisons and proficiency testing schemes. The document emphasizes that laboratories must have quality control procedures to monitor validity of tests and ensure trends in results are detectable.
Please Subscribe to this Channel for more solutions and lectures
http://www.youtube.com/onlineteaching
Chapter 9: Inferences from Two Samples
9.2: Two Means, Independent Samples
This document provides information about transfusion transmissible infection (TTI) testing performed at a laboratory. It describes 5 tests conducted - for HIV, HBV, HCV, syphilis, and malaria. The methods used are ELISA, MEIA, rapid tests, TPHA for syphilis, and slide testing for malaria. Detailed procedures are provided for each test, including sample preparation, controls, interpretation of results, and any special equipment used like the Miniswift or AXSym machines for automated ELISA and MEIA.
This document discusses various special stains used in pathology to identify structures in tissue samples. It provides details on stains for lipids, carbohydrates, iron, myeloperoxidase, alkaline phosphatase, reticulin fibers, and other components. The stains discussed include Sudan Black B, Periodic Acid Schiff, Perl's Iron stain, myeloperoxidase, and reticulin stain. The objectives of special staining are to provide a definitive diagnosis and aid in differential diagnoses of hematological conditions.
IMPLEMENTATION OF QUALITY CONTROL PERFORMANCE CRITERIA AND APPROVED GUIDELINE...Moustafa Rezk
IMPLEMENTATION OF QUALITY CONTROL PERFORMANCE CRITERIA AND APPROVED GUIDELINES FOR UPGRADING OF CLINICAL CHEMISTRY LABORATORY PROCEDURES IN ALEXANDRIA UNIVERSITY HOSPITALS
This document provides information on performing and interpreting a gram stain of sputum samples. It describes the principles behind gram staining, outlines the staining procedure and sources of errors. It also details how to evaluate sputum quality, quantify and identify bacteria, fungi and other cells seen on gram stain. Quality control measures are highlighted to help ensure accurate results.
The document discusses Chi-Square tests, which are used when assumptions of normality are violated. It provides requirements for Chi-Square tests, including that variables must be independent and samples sufficiently large. The key steps are outlined: determine appropriate test, establish significance level, formulate hypotheses, calculate test statistic using frequencies, determine degrees of freedom, and compare to critical value. An example compares party membership to opinions on gun control to demonstrate a Chi-Square test of independence.
ISO 15189 is an international standard that specifies the general requirements for the competence of medical laboratories. It is based on ISO 17025 for testing and calibration laboratories and ISO 9001 for quality management systems. ISO 15189 has both management and technical requirements that medical laboratories must meet in order to be accredited. The standard is designed to ensure that laboratories consistently deliver accurate, reliable and timely medical testing services.
The document describes developing an alternative diagnostic test for detecting the severe strain of Leuser virus based on measuring levels of enzyme X in serum. The researchers optimized an enzyme assay to measure enzyme X levels, determining the best pH, temperature, measurement time and wavelength. They established a cutoff level using samples from patient set A and then classified unknown samples from patient set B as mild or severe strain based on this cutoff. The assay demonstrated 100% sensitivity and 70% specificity. The enzyme X assay shows potential as a rapid, specific and cheaper alternative diagnostic test for the severe strain of Leuser virus.
Validation of Opsonophagocytic assay for pnemococcal vaccinekishor kulkarni
This document describes the validation of an opsonophagocytic killing assay for Streptococcus pneumoniae. The objectives of the validation study were to assess the assay's precision, linearity, accuracy, robustness, and specificity according to ICH guidelines. The results showed that the assay had 100% precision when testing multiple samples, good linearity with correlation coefficients between 0.948-1.000, 100% accuracy when compared to a reference standard, was robust to changes in effector-to-target cell ratios up to 100:1, and demonstrated specificity by inhibiting ≥80% of opsonophagocytic activity with homologous polysaccharides and ≤20% inhibition with heterologous polysaccharides. The conclusion is
This document discusses different types of ELISA results - quantitative, qualitative, and semi-quantitative. It provides details on generating a standard curve for quantitative ELISAs, including examples of pipetting schemes and plotting absorbance values versus analyte concentration. For qualitative ELISAs, it describes comparing sample absorbance values to a cut-off calibrator. The document also gives an example of calculating a cut-off value and criteria for interpreting qualitative ELISA results.
The job market is difficult, but you have managed to obtain a temporary job in a medical research team. You are pleasantly surprised to be told that you were appointed because of your expertise in statistics. The team is investigating the effects of poisonous metals on human health. They will be obtaining data from various sources, but in the meantime they have given you some old data for you to learn about the kinds of analysis required.
The main purpose of this research project was to develop a quantitative serological diagnostic enzyme assay to detect and differentiate between the two strains of Yellow Eye Disease. The research optimized an enzyme assay that achieved a 97.5% diagnostic success rate compared to the current ELISA method. Investigation into the pathogenesis through urinalysis, haemolysis assays, and H&E staining indicated hepatic malfunction and haemolysis in diseased patients. The results provide a foundation to improve both treatment and diagnosis of the more severe strain two of the virus.
This document describes the development and validation of an assay to measure vitamin K1 (phylloquinone) levels in human serum using liquid chromatography-tandem mass spectrometry. The assay was found to have acceptable precision, linearity, recovery, accuracy and specimen stability. No ion suppression or interference from other compounds was demonstrated. The method provides a reliable means for analyzing vitamin K1 levels in serum.
LC-MS/MS analysis of emerging food contaminantsSCIEX
Recently (November 2014), threats in the form of letters were sent to farming and dairy industry leaders in New Zealand. The letters were accompanied by small packages of milk powder that were shown to contain a concentrated form of the pesticide 1080 (sodium fluoroacetate). The sender demanded that the New Zealand government stop using 1080 for pest control. Sodium fluoroacetate is used to protect New Zealand’s native flora and fauna against introduced pests like possums and ferrets. Opponents, however, argue that it also kills native animals and contaminates the environment.1-2
Such criminal threats are a potential danger and weaken consumers’ trust in the food supply chain. Accurate and reliable analytical methods are needed to monitor food ingredients and final products to ensure food safety in light of this threat.
Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is an ideal analytical technique to detect polar analytes in complex food samples.
Here we present first results of method development to detect sodium fluoroacetate in milk and infant formula. The sample preparation protocol consists of a simple acetonitrile extraction and defatting using hexane. LC separation was achieved using a HILIC column in normal phase mode. The mass spectrometer was operated in Multiple Reaction Monitoring (MRM) mode. In MRM mode the transition of a molecular ion into a characteristic fragment ion is monitored. The monitoring of more than a single fragment ion allows not only quantitation but also highly confident identification based on the ratio between quantifier and qualifier transitions.
Initial studies show that sodium fluoroacetate can be detected at concentrations below 1 ng/mL (below 10 ng/mL in matrix) using the SCIEX QTRAP® 4500 system, with good accuracy and repeatability. Linearity for quantitation was achieved over 3 orders of magnitude (0.1 to 100 ng/mL). Future experiments are planned to further increase sensitivity, simplify sample preparation and to include an internal standard to correct low recoveries and matrix effects.
ReaPanMicro is a diagnostic test that uses nucleic acid detection to confirm the absence of bacteria in urine samples within 15 minutes. It has a high sensitivity of nearly 100% negative predictive value. The test involves filtering urine and staining it with reagents before running it on a flow cytometer. Clinical trials on over 250 urine samples showed the test accurately identified samples as positive or negative compared to culture results, establishing a threshold of 3000 counts to determine negativity. The portable and standardized test can accurately rule out bacterial infections in point-of-care settings.
The document describes a method for quantifying several amphetamines (amphetamine, methamphetamine, MDMA, ephedrine, pseudoephedrine) in human blood and serum samples using UPLC-MS/MS. A basic mobile phase was used contrary to conventional practice, improving analyte retention, resolution, and sensitivity. The method involved protein precipitation extraction, UPLC separation with a basic mobile phase in 3.5 minutes, and mass spectrometric detection. Validation experiments demonstrated the method was rapid, robust, and suitable for forensic toxicology applications.
What is Nephelometry,and a fully automated Nephlometer analyzer for protein a...Shahid Nawaz
Nephelometry is commonly used to determine protein levels in body fluids like serum and urine. It works by measuring the light scattered by antigen-antibody complexes formed when a sample containing antigen is mixed with corresponding antiserum. The amount of light scattered is proportional to the antigen concentration in the sample. This document provides instructions for using a nephelometer to test samples for C-reactive protein (CRP) levels, including preparing reagents and standards, running samples, generating a calibration curve from standards, and recording results. CRP levels between 0.0-1.0 mg/dL are considered normal.
Poster demonstrating the results from the development/verification project for the quantitation of all- trans retinol and alpha tocopherol in human serum.
This document compares three commercially available enzyme-linked immunosorbent assays (ELISAs) for detecting immunoglobulin G antibodies to tetanus toxoid. Two international reference standards were tested in quadruplicate using each ELISA kit. The Binding Site ELISA provided results most closely matching the reference standards, while the Scimedx ELISA consistently reported lower results and the Euroimmun ELISA higher results. When testing 83 serum samples, the ELISAs showed 78% agreement using manufacturers' cutoffs. The Binding Site ELISA identified the fewest samples (3) as non-protective, compared to 19 for the Scimedx ELISA and 6 for the Euroimmun ELISA. Accurate tetanus antibody testing is important for
This document summarizes the validation of a quantitative LC/MSMS method to measure the cleavage of a 17-residue SNAP-25 epitope by botulinum neurotoxin serotype A (BoNT/A). Peptides representing the intact 17-mer epitope and its cleavage products (11-mer and 6-mer) were characterized. The method was validated and used to evaluate the kinetics of 17-mer cleavage, finding near-completion at 40-45 minutes. The validated method will be applied to evaluate potential BoNT/A inhibitors and detect contamination in samples.
Benzonase® endonuclease: use and clearance with a TFF step in a cell culture-...Frédéric Sengler
Benzonase® endonuclease is a powerful tool for degrading all
forms of (deoxy)ribonucleic acids (DNA/RNA) in cell culture
harvests to base pair (BP) lengths under 8 units. It is effective
over a wide range of conditions including temperature, pH, and
varying concentrations of Mg2+, detergents, chelating agents,
and monovalent cations. It is often employed in the production
of viral vaccines, completely digesting DNA and RNA to improve
clearance and reduce solution viscosity.
Removing Benzonase® endonuclease, a 60 kD dimer, from the
process stream post-treatment may be achieved with Tangential
Flow Filtration (TFF) with the appropriate molecular weight
cut-off membrane (MWCO), typically 300 kD. This process has
heretofore not been well described in the literature, so the
present study fills this gap pairing Benzonase® endonuclease
treatment of a DNA-spiked inactivated flu virus cell culture
harvest with Pellicon® 2 cassettes, for clearance of the digested
DNA and remaining Benzonase® endonuclease by diafiltration.
Automated SPE for Capillary Microsampling PosterRick Youngblood
1) An automated SPE method using small single-use cartridges was evaluated for plasma samples derived from capillary microsampling as an alternative to manual protein precipitation.
2) Results from the automated SPE method were comparable to manual protein precipitation in terms of accuracy, precision, calibration curves, and limits of quantification. The automated method provided equivalent results but with less hands-on time.
3) Further optimization is possible, including reducing the plasma sample volume and elution volume to allow analysis of samples from microsampling techniques using minimal volumes.
The document discusses the state of coagulation testing and summarizes key findings from surveys of clinical coagulation laboratories. It finds that (1) screening tests like APTT and PT have limitations in detecting low titer lupus anticoagulants, (2) there is significant variability between laboratories in factor assays due to differences in reagents and methods, and (3) further standardization could help improve the performance and interpretation of coagulation tests.
This document summarizes a study evaluating the utility of using multiplex panels of biomarkers for screening purposes without prior knowledge of biomarkers of interest. Fifteen multiplex panels were developed containing up to ten assays each, requiring less than 1 mL of sample to measure 122 biomarkers total. Assays showed low cross-reactivity, broad dynamic ranges, and good reproducibility. The multiplex panels allowed rapid screening and stratification of patient populations to identify potential biomarker targets.
A large amount of data is available on the functional impact of missense mutations in TP53 tumor suppressor gene. and on mutation patterns in many different cancers. TP53 direct sequencing is a time-consuming method with limitations in detection level. Here we describe the development and verification of a TP53 Next Generation Sequencing method based on Ion AmpliSeq technology. Preliminary data demonstrate that the TP53 Ion AmpliSeq panel and Ion Reporter Software analysis solution meets the requirements of clinical research laboratories.
This document compares the efficiency of using automated solid phase extraction coupled with high performance liquid chromatography and tandem mass spectrometry (SPE-HPLC/MS/MS) versus traditional immunoassay screening followed by SPE-GC/MS or SPE-LC/MS/MS confirmation for analyzing postmortem blood samples submitted in death investigation cases. The study finds that automated SPE-HPLC/MS/MS using an Instrument Top Sample Preparation (ITSP) system provides results generally in agreement with traditional methods, with improved efficiency through integrated online sample preparation and reduced analysis time and costs. The automated method was able to extract and analyze samples stored for up to 12 months, demonstrating its robustness for difficult biological matrices.
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2. • Leuser Virus has re emerged in South East Asia.
• Along with the historically mild strain 1, a second fatal strain is ravaging the
rural communities.
• The current standard for testing is an ELISA-based blood test, however it is
unable to differentiate this second fatal strain.
• Early research has shown that the second strain only, causes yellowing of
the eyes and an increase in enzyme X serum levels.
• Consequently we have developed an assay for this enzyme, to improve
diagnostics and allow for differentiation of the two strains, which will
prevent the unnecessary and costly treatment of strain 1.
• Samples from a deceased patient with strain 2 have also been investigated
to find out more about the pathology of the virus, however this
presentation will focus on the development of the assay only, including:
• Optimisation of parameters
• Determination of cut off points and performance characteristics
• Comparison with the current standard of testing (ELISA)
Introduction
4. Assay optimisation
• Jon Darkrows testing unknown enzymes simulation
programme was used to determine the optimal pH,
temperature and incubation time of the assay method.
• Each parameter was investigated individually, by keeping
the other parameters constant and observing the
relationship between changing the parameter and
changes in enzyme activity
Fig (I) John Darkrows testing unknown enzymes simulation programme.
Optimal pH -
• Assay ran in triplicates Physiological
temperature (37C)
• 500mg/dL substrate concentration
• Integer intervals between pH 2 and pH
12
Optimal temperature –
• Assay ran in triplicates
• Optimal pH as previously
determined
• 500 mg/dL substrate concentration
• 5C intervals between 15C and
55C
Data used to
confirm the optimal
assay duration for
the remaining assay
runs.
Data used to
determine the
optimal assay
duration for
the remaining
assay runs
5. Serum samples from previously diagnosed Patient set A were ran on
the optimised assay to produce triplicate absorbance values:
1. A solution of 0.1 M Tris HCl pH 8 assay buffer and 10mM enzyme X
substrate was prepared
2. 1ml was added to 20 test tubes, each labelled with a set A patient,
A1-A20, which were placed in a water bath at 40oC for five minutes
3. 0.1 ml of sample serum from each patient was added to it’s
labelled test tube and the tubes were left to incubate for 10
minutes.
4. The tubes were then removed from the water bath and 1ml sodium
hydroxide was added immediately and mixed.
5. Absorbance of each test tube was read at the optimal wavelength
(which couldn’t be optimised for) and recorded
Determination of clinical cut off points
Figure (III) Samples were incubated in a 40C
water bath. Image taken from www.jsr.kr.
Figure (II) Patient serum sample.
6. • To calculate the enzyme concentration a standard curve of
enzyme X concentration against assay absorbance was
made from research data, and this was used to convert the
absorbances.
• Possible cut off points were determined at different
enzyme X concentrations that were higher than the
majority seen in strain 1 patient samples (A1-A10) and
lower than the majority seen in strain 2 patient samples
(A11-A20).
• 1ml serum samples from undiagnosed patient set B were
ran on the assay as previously described to determine their
enzyme x concentrations
• Diagnosed with the different cut off points.
• These diagnosis's were compared to the known diagnosis of
each patient, to calculate the specificity and sensitivity of
each cut off point.
• For this presentation only the chosen cut off point will be
discussed.
y = 0.2108x + 0.4198
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
Absorbance
(Au)
Enzyme X concentration (mg/L)
Standard curve of enzyme X concentration
against absorbance
Fig (IV) Standard curve of enzyme X concentration against
absorbance. Used to convert Set A sample absorbances.
7. Comparison with the ELISA standard
As the ELISA is the current standard test for the diagnosis of Leuser Virus, serum samples from Patient Sample
Set C were tested using the enzyme X assay method, as previously described, and the ELISA method, as follows,
for comparison.
1. 100 ml of capture antibody was transferred to each well, incubated for a week
at 4C and removed from by inverting, washing with 300 ml wash buffer and
blotting the wells three times.
2. 300 ml blocking buffer was added to each well, incubated at room temperature
for an hour, then removed as previously described.
3. 100 ml of a negative control sample not containing Leuser virus antigen,
positive control sample containing the antigen, reference sample, patient C1
sample, patient C2 sample were added to labelled wells, incubated for an hour
at room temperature, then removed as previously described.
4. This was repeated with detection antibody
5. 100 ml streptavidin-HRP, diluted 1:40 in reagent dilutent, was transferred to each well, incubated for 20 minutes at room
temperature and removed as previously described.
6. 100 ml of mixed 1:1 substrate solution A to B was added to each well and incubated for 20 minutes
7. The reaction was stopped by adding 50 µL of stop solution.
8. The plates were then read at 450 nm in a plate reader.
Fig (V) 96 well plate for ELISA.
9. Enzyme optimisation
Fig (VI) Graph showing the activity of enzyme X at pH2-pH7
0.00
100.00
200.00
300.00
400.00
500.00
600.00
2 3 4 5 6 7 8 9 10 11 12
Enzyme
X
Concentration
(mg/dL)
pH
Effect of pH on Enzyme X activity
Optimal pH was
found to be pH 8
10. Enzyme optimisation
Fig (VII) Graph showing the activity of enzyme X between 15-55 C
0.00
100.00
200.00
300.00
400.00
500.00
600.00
2 3 4 5 6 7 8 9 10 11 12
Average
Poduct
Concentration
(mg/dL)
pH
Effect of pH on Enzyme X activity
0.00
100.00
200.00
300.00
400.00
500.00
600.00
15 20 25 30 35 40 45 50 55
Average
Product
Concentration
(mg/dL)
Temperature (C)
Effect of temperature on Enzyme X activity
40
Optimal temperature was found to be 40C
Fig (VI) Graph showing the activity of enzyme X at pH2-pH7
11. Enzyme optimisation
0.00
100.00
200.00
300.00
400.00
500.00
600.00
2 3 4 5 6 7 8 9 10 11 12
Average
Poduct
Concentration
(mg/dL)
pH
Effect of pH on Enzyme X activity
0.00
100.00
200.00
300.00
400.00
500.00
600.00
15 20 25 30 35 40 45 50 55
Average
Product
Concentration
(mg/dL)
Temperature (C)
Effect of temperature on Enzyme X activity
Fig (VIII) Enzyme simulation programme runs for pH optimisation at
physiological (37oC) temperature and 500 mg/dL substrate concentration.
Optimal
incubation
duration
was found
to be 10
minutes
Fig (VII) Graph showing the activity of enzyme X between 15-55 C.
Fig (VI) Graph showing the activity of enzyme X at pH2-pH7.
Fig (IX) Enzyme simulation programme runs for temperature
optimisation at pH 8 and 500 mg/dL substrate concentration.
12. Determination of cut-off points and
performance characteristics
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
4.20
4.40
4.60
4.80
5.00
5.20
5.40
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Enzyme
X
Concentration
(mg/L)
Pateint Number
Concentration of enzyme X in Set A Patient
Samples
Figure (x) Graph showing the determination of the clinical cut off point from
patient set A sample enzyme concentrations.
Table (I) Comparison of patient set B sample assay and confirmed
diagnosis’s used to calculate specificity and sensitivity.
The enzyme X
concentration that was
selected as the clinical cut
off value for the assay was
2.30 mg/dL, as it gave the
highest sensitivity of 100%,
and a relatively high
specificity of 90%.
SampleLabel
Assay Diagnosis (Strain) Confirmed Diagnosis
(Strain)
TP/FP/TN/FN
Patient B1 2 2 TP
Patient B2 2 1 FP
Patient B3 1 1 TN
Patient B4 2 2 TP
Patient B5 2 2 TP
Patient B6 1 1 TN
Patient B7 1 1 TN
Patient B8 2 2 TP
Patient B9 2 2 TP
Patient B10 1 1 TN
Patient B11 2 2 TP
Patient B12 2 2 TP
Patient B13 1 1 TN
Patient B14 1 1 TN
Patient B15 2 2 TP
Patient B16 1 1 TN
Patient B17 2 2 TP
Patient B18 2 2 TP
Patient B19 1 1 TN
Patient B20 2 2 TP
Patient B21 1 1 TN
Patient B22 2 2 TP
Patient B23 2 2 TP
Patient B24 1 1 TN
Patient B25 2 1 FP
Patient B26 2 2 TP
Patient B27 1 1 TN
Patient B28 2 2 TP
Patient B29 2 2 TP
Patient B30 1 1 TN
Patient B31 2 2 TP
Patient B32 1 1 TN
Patient B33 2 2 TP
Patient B34 1 1 TN
Patient B35 1 1 TN
Patient B36 1 1 TN
Patient B37 2 2 TP
Patient B38 1 1 TN
Patient B39 1 1 TN
Patient B40 2 2 TP
Assay cut
off point
2.30 mg/dL
Sensitivity calculation –
20/20 x 100
Sensitivity = 100 %
Specificity calculation –
18/20 x 100
Specificity = 90%
13. ELISA Enzyme X Assay
Average Patient C1 Result 0.748 AU at 450 nm 4.90 mg/dL Enzyme X concentration
Standard Deviation +/- 1.27 x 10 ֿ ² AU +/- 5.90 x 10 ֿ ² mg/dL
Clinical cut off value 0.808 AU at 450 nm 2.30 mg/dL Enzyme X concentration
Diagnosis (Negative, Positive – strain
undifferentiated, Strain 1, Strain 2)
Negative Strain 2
ELISA Enzyme X Assay
Average Patient C2 Result 1.13 AU at 450 nm 1.10 mg/dL Enzyme X concentration
Standard Deviation +/- 4.81 x 10 ֿ ² AU +/- 1.48 x 10 ֿ ¹ mg/dL
Clinical cut off value 0.808 AU at 450 nm 2.30 mg/dL Enzyme X concentration
Diagnosis (Negative, Positive - strain
undifferentiated, Strain 1, Strain 2)
Positive – strain undifferentiated Strain 1
Comparison to the ELISA standard
Table (II) – Comparison of ELISA for patient C1 diagnosis and Enzyme X Assay for diagnosis.
Table (III) – Comparison of ELISA for patient C2 diagnosis and Enzyme X Assay for diagnosis.
14. Conclusions
• In summary, this work provides proof of principle to validate a full scale clinical evaluation of the
enzyme X assay for the diagnosis of ‘yellow eye’ Leuser Virus.
• The parameters of the assay for this investigation should be kept to a pH of 8, temperature of 40oC
and an incubation time of 10 minute. Further investigation is needed to determine the optimal
wavelength of this assay, as the clinical investigation was limited by its inability to determine this.
• The clinical cut off point of this assay should be kept at 2.30 mg/dL enzyme X concentration to insure
100% sensitivity and keep specificity as high as possible.
• The new assay overcomes important barriers in the diagnosis of the yellow eye strain, currently
limited by the standard ELISA test, which is not able to differentiate between this and strain 1.
• Although the ELISA was found to be more specific, the 100% sensitivity of the new assay is highly
desired for diagnostics, as ‘yellow eye’ is fatal and so a false negative result will lead to the patients
death as they will not receive vital treatment.
• Further investigation is needed to confirm either the ELISA, negative, or Enzyme X Assay, strain 2,
diagnosis of patient C1, in order to calculate specificity and sensitivity of the ELISA in diagnosing
undifferentiated Leuser Virus for comparison.