Improved Method for the ASTM Platelet and Leukocyte Assay: Use of Minimal Heparinization in a Screening Test for Hemocompatibility of Blood-Contacting Medical Devices

2018 ◽  
Vol 13 (1) ◽  
Author(s):  
Tim Schatz ◽  
Sarah Howard ◽  
Deanna Porter ◽  
Kent Grove ◽  
Mark E. Smith ◽  
...  
Keyword(s):  
Medical Devices ◽  
High Density Polyethylene ◽  
Complete Blood Count ◽  
Clinical History ◽  
Positive Control ◽  
Final Concentration ◽  
Surface Adhesion ◽  
Improved Method ◽  
Robust Response ◽  
Positive Controls

Most blood-contacting medical devices must be assessed for potential thrombogenicity prior to regulatory approval. A common assay for screening and qualifying devices involves monitoring the reduction of platelet and leukocyte (P&L) counts in whole blood exposed to the device. We have validated an improved method for assessing a device's effect on platelet activation and surface adhesion, offering significant improvement over the current ASTM F2888-13 method, which uses blood fully anticoagulated by acidified citrate (known to significantly inhibit platelet responsiveness). Our method uses minimal heparinization (final concentration 1 IU/mL) to optimize the response to commonly used control materials: latex, black rubber, and high-density polyethylene (HDPE). We also have shown the assay's capacity to appropriately assess a legally marketed comparator device (LMCD) with a documented clinical history. The test materials were prepared for incubation and allowed to remain in contact with the citrated or heparinized blood for ∼1 h at 37 °C. A complete blood count was performed prior to exposure, and at the end of the incubation period, reductions in P&L counts were recorded. Results from citrate-anticoagulated assay showed only a marginal response to the positive control, black rubber. Using heparinized blood, the assay generated a robust response to the positive controls, the “intermediate scoring” controls, and also assessed a legally marketed and approved device as clearly nonthrombogenic. This modification adds robustness and sensitivity to this quick and inexpensive thrombogenicity assay and should be incorporated into the next ASTM standards.

scholarly journals Reduction in the Number of Animals and the Evaluation Period for the Positive Control Group in Tg.rasH2 Short-Term Carcinogenicity Studies

2012 ◽  
Vol 31 (5) ◽  
pp. 423-429 ◽  
Author(s):  
Sudhir A. Shah ◽  
Madhav. G. Paranjpe ◽  
Philip I. Atkins ◽  
Eias A. Zahalka
Keyword(s):  
Positive Control ◽  
Lung Tumors ◽  
Control Group ◽  
Short Term ◽  
Evaluation Period ◽  
Target Organs ◽  
The Us ◽  
Assessment Committee ◽  
Robust Response ◽  
Positive Controls

The lack of a clear guidance on the adequate number of animals used for positive controls in the short-term (26-weeks) transgenic mouse carcinogenicity studies has resulted in the use of high number of animals. In our earlier Tg.rasH2 studies, 25 mice/sex were used in the urethane-positive control dose groups that were sacrificed by 18 weeks. Based on a robust response, several of our protocols for Tg.rasH2 studies with 15 mice/sex and terminal sacrifice at 17 ± 1 weeks were submitted and accepted by the Carcinogenicity Assessment Committee of the US Food and Drug Administration since we demonstrated close to 100% response for the development of lung and splenic tumors (target organs) in 500 mice/sex. These 500 mice/sex included 17 groups of 25 mice/sex and 5 groups of 15 mice/sex.  The objective of this investigation was to determine whether the number of animals can be further reduced along with the shortened duration of exposure to urethane. Accordingly, 10 Tg.rasH2 mice/sex/group were administered a total of 3 intraperitoneal (IP) injections of urethane (1000 mg/kg per day) on study days 1, 3, and 5, and the presence of tumors in the lungs and spleen was evaluated after 8, 10, 12, 14, or 16 weeks. Our results demonstrate that 100% of the mice at 8 weeks had developed lung tumors, whereas close to 100% of the mice at 14 weeks had developed splenic tumors. Based on the development of lung tumors alone in 100% of the mice, we recommend that 10 mice/sex are sufficient and that these mice can also be sacrificed as early as 10 ± 1 weeks following the administration of urethane.

Solutions and gels containing a sugarcane-derived cystatin (CaneCPI-5) reduce enamel and dentin erosion in vitro

2021 ◽  
Author(s):  
Lethycia Almeida Santos ◽  
Tatiana Martini ◽  
João Victor Frazão Câmara ◽  
Fabiana Navas Reis ◽  
Adriana de Cássia Ortiz ◽  
...  
Keyword(s):  
Artificial Saliva ◽  
Dental Erosion ◽  
Positive Control ◽  
New Approach ◽  
Enamel Erosion ◽  
Ph Cycling ◽  
Bovine Enamel ◽  
Dentin Erosion ◽  
Positive Controls

The effect of solutions and gels containing a sugarcane-derived cystatin (CaneCPI-5) on the protection against enamel and dentin erosion in vitro was evaluated. Bovine enamel and dentin specimens were divided into two groups (n=135 and 153/group for enamel and dentin, respectively) that were treated with solutions or chitosan gels containing 0.1 or 0.25 mg/ml CaneCPI-5. The positive controls for solutions and gels were Elmex Erosion Protection™ solution and NaF gel (12,300 ppm F), respectively. Deionized water and chitosan gel served as controls, respectively. The solutions were first applied on the specimens for 1 min and the gels for 4 min. Stimulated saliva was collected from 3 donors and used to form a 2 h acquired pellicle on the specimens. Then, the specimens were submitted to an erosive pH cycling protocol 4 times/day for 7 days (0.1% citric acid pH 2.5/90s, artificial saliva/2h, artificial saliva overnight). The solutions and gels were applied again during pH cycling, 2 times/day for 1 min and 4 min, respectively, after the first and last erosive challenges. Enamel and dentin losses (µm) were assessed by contact profilometry. Data were analyzed by 2-way ANOVA and Tukey´s test (p <0.05). All the treatments significantly reduced enamel and dentin loss in comparison with controls. Both CaneCPI-5 concentrations had a similar protective effect against enamel erosion, but only the higher concentration was as effective against dentin erosion as the positive control. Regarding the vehicles, only the 0.1 mg/ml gel performed worse than the positive control for dentin. CaneCPI-5 reduced enamel and dentin erosion to a similar extent as the fluoride-containing vehicles. However, dentin requires higher CaneCPI-5 concentrations, in the case of gels. Solutions or gels containing CaneCPI-5 might be a new approach to protect against dental erosion.

Preparation of His-Tagged Armored RNA particles as positive controls for Real-Time RT-PCR Detection of Zika virus

2021 ◽  
Vol 31 (4) ◽  
pp. 18-27
Author(s):  
Do Thi Quynh Nga ◽  
Ha Thi Phuong Mai ◽  
Tran Thi Hai Au ◽  
Vu Thi Kim Lien ◽  
Vu Thi Bich Hau ◽  
...  
Keyword(s):  
Real Time ◽  
Zika Virus ◽  
Room Temperature ◽  
Virus Detection ◽  
Positive Control ◽  
Reverse Transcription Pcr ◽  
His Tag ◽  
Armored Rna ◽  
Genomic Regions ◽  
Positive Controls

Armored RNA (AR) is a good candidate for creating nuclease-resistant RNA positive controls in the nucleic acid - based assay for RNA viruses. To simplify the production and purifcation of armored RNA, a single plasmid double – expressing His6-tag system was designed. His-tag armored RNA particles were purifed using his-tag affnity. A genomic fragment of the zika virus consisting of the encoding sequences of flavi-M, flavi-E-C protein targeted for zika virus was selected to prepare a positive control. In this study, we have successfully produced His-taged MS2- phage like particles carrying specifc genomic regions (M and E genes) to monitor the procedures of real-time Reverse transcription-PCR for Zika virus detection in one plasmid double expression. AR-ZIKA is completely resistant to DNase and RNase, stable in normal human EDTA plasma at room temperature for at least 60 and 15 days at 40C and room temperature respectively.

Connecting Across Competencies: Leveraging Best Practices for Processing

2021 ◽  
Vol 55 (s3) ◽  
pp. 6-11
Author(s):  
Terra A. Kremer ◽  
Kaumudi Kulkarni ◽  
Christopher Ratanski ◽  
Lorraine Floyd ◽  
Christopher Anderson
Keyword(s):  
Best Practices ◽  
Medical Devices ◽  
Technical Information ◽  
Test Sample ◽  
Positive Control ◽  
Standard Test ◽  
Test Method ◽  
Cleaning Validation ◽  
Validation Parameters ◽  
Method Suitability

Abstract The AAMI working group ST/WG 93 is finalizing a standard (AAMI ST98) for the cleaning validation of reusable medical devices based on guidance from the technical information report AAMI TIR30:2011/(R)2016. A number of analytical best practices are being considered for this new standard. Test method suitability for processing cleaning validations historically has been established using one positive control and performing an extraction efficiency. The new cleaning validation standard is proposed to require a change from only one replicate test sample to three when performing method suitability. This change will affect manufacturers; therefore, the value of and consideration for performing these additional replicates requires explanation. This article discusses how variation of validation parameters can affect the accuracy and precision during method suitability testing. Multiple replicates are needed to understand the variability of method extraction and impact on cleaning validations of reusable medical devices.

scholarly journals Impact of raw cocoa and chocolate on lipid profile and liver function in alcohol-induced toxicity Wister rats

2017 ◽  
Vol 5 (2) ◽  
pp. 254
Author(s):  
Alaa Saleh ◽  
Murwan Sabahelkhier
Keyword(s):  
Fatty Liver ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Treated Group ◽  
Positive Control ◽  
Alcoholic Fatty Liver ◽  
Alcohol Group ◽  
High Doses ◽  
The Impact ◽  
Positive Controls

The aim of study is to evaluate the impact of raw cocoa and processed cocoa (galaxy chocolate) in the treatment of alcoholic fatty liver compare to prednisolone drug as common medication to treat fatty liver and carried out in Al-Neelain University in 2016. Eighteen female Wister rats were classified into six groups: Group one represented negative (H2O), Group two positive controls (0.5 ml/40% alcohol), Group three represented doses of 0.67g\kg raw cocoa, Group four represented dose of 1.35 g/kg raw cocoa, Group five represent dose of 5.4 g/kg galaxy chocolate, Group six received dose of 4.8 mg/kg prednisolone. The results show that the cocoa with low and high doses, galaxy chocolate and prednisolone significantly reduce the ALT, AST and GGT, and a significant decrease in the ALT level after administration of prednisolone drug when compared high cocoa dose group. Whereas, Galaxy chocolate significantly reduce the AST compared to low dose cocoa treated group. Also there is a significant increase in HDL and decrease in Total cholesterol, Triglycerol and low density lipoprotein in all treated groups compared to positive control. The ratio between Triglycerol: HDL and LDL: HDL show a significant reduction in group treated with galaxy chocolate compared to other treated groups. The results concluded that, cocoa in both forms raw and processed are efficient in the treatment of alcoholic fatty liver in comparison with prednisolone drug.

scholarly journals DEVELOPMENT OF RECOMBINANT POSITIVE CONTROL FOR AFRICAN SWINE FEVER VIRUS PCR DETECTION

2020 ◽  
Vol 13 (6) ◽  
pp. 58-63
Author(s):  
M. Kit ◽  
Keyword(s):  
African Swine Fever Virus ◽  
Animal Health ◽  
African Swine Fever ◽  
Positive Control ◽  
Fever Virus ◽  
Laboratory Diagnostics ◽  
E Coli ◽  
Pcr Assays ◽  
World Organisation ◽  
Positive Controls

Recombinant plasmids containing target sequences are widely used as positive controls for PCR laboratory diagnostics. The aim of the study was development of recombinant positive control containing a fragment of B646L gene of African swine fever virus. The sequence of interest encodes targets of all the PCR assays for African swine fever laboratory diagnostics recommended by World Organisation for Animal Health. A plasmid containing 1763 bp insertion was cloned in E .coli DH5α strain. After purification, the plasmid ten-fold serial dulutions were used as a positive control while PRC testing. A minimal detectable copy number was 20 copies per reaction for both conventional and real-time PCR assays. The developed plasmid could be used as a safe and effective positive control while ASF laboratory diagnostics by PCR.

scholarly journals PRIMARY SLX TEST USING REAL-TIME PCR BASED ON HIGH RESOLUTION MELTING (HRM) ON MICROFILARIA EXAMINATION

2021 ◽  
Vol 5 (1) ◽  
pp. 26
Author(s):  
Bagus Muhammad Ihsan ◽  
Cecep Dani Sucipto ◽  
Khayan Khayan
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Wuchereria Bancrofti ◽  
Positive Control ◽  
Amplification Curve ◽  
Ct Value ◽  
Polymerase Chain ◽  
Positive Controls

Background: Filariasis patients can be a source of transmission if their blood still contains microfilariae. One of the Polymerase Chain Reaction (PCR) methods used is High Resolution Melting (HRM), using primary specificity testing. Purpose: To test the specificity of SLX primer. The samples used for this test were isolates of Salmonella., Klebsiella, Pseudomonas, negative and positive controls for Brugia malayi and Wuchereria bancrofti. Method: The design in this study is a quasi-experiment by testing the specificity of SLX primer using HRMbased real-time PCR based on the Cycle Threshold (CT) value observed through the amplification curve. Result: The real-time PCR results showed that no CT was released in the bacterial samples, and there was a CT value in the positive control. The results of this study indicate that specific SLX primer can be used in identifying microfilariae. Conclusion: SLX primer have a reasonable specificity because they cannot detect the existence of microorganisms in the samples other than microfilariae.

Improved assay for alcohol dehydrogenase activity in serum by centrifugal analysis.

1984 ◽  
Vol 30 (11) ◽  
pp. 1817-1820 ◽  
Author(s):  
S Kato ◽  
H Ishii ◽  
S Kano ◽  
S Hagihara ◽  
T Todoroki ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Clinical Utility ◽  
Final Concentration ◽  
Dilution Curve ◽  
Healthy Individuals ◽  
Improved Method ◽  
Simultaneous Reduction ◽  
Alcohol Dehydrogenase Activity

Abstract We describe an improved method for determination of alcohol dehydrogenase (EC 1.1.1.1) activity in 60 microL of human serum, based on conversion of ethanol to acetaldehyde with simultaneous reduction of NAD+ in glycine NaOH buffer (pH 9.0) at 37 degrees C in a centrifugal analyzer. The final concentration of NAD+ was 10 mmol/L and ethanol was 20 mmol/L. The dilution curve was linear with enzyme activity up to 200 U/L, and results by this method correlated well with those by a manual method (N Engl J Med 279: 241-248, 1968). Within-run precision (CV) was 0.9 to 8.2% over the range of 4.5 to 88.1 U/L, and day-to-day precision was 5.4 to 5.6%. In sera from 198 healthy individuals, mean alcohol dehydrogenase activity was 1.6 (SD 1.2, range 0-5) U/L. To evaluate the clinical utility of determining alcohol dehydrogenase, we measured the activity of alanine aminotransferase and alcohol dehydrogenase in sera from 470 patients with various diseases in our hospital, and found that results for the two enzymes did not correlate well.

scholarly journals Sensitivity of polymerase chain reaction for detection of known aliquots of Trypanosoma cruzi in the blood of mice: an in vitro study

2002 ◽  
Vol 35 (5) ◽  
pp. 487-490 ◽  
Author(s):  
Rozália F. Campos ◽  
Juracy B. Magalhães ◽  
Eliana A.G. Reis ◽  
Mitermayer G. Reis ◽  
Sonia G. Andrade
Keyword(s):  
Polymerase Chain Reaction ◽  
Trypanosoma Cruzi ◽  
In Vitro Study ◽  
High Sensitivity ◽  
Positive Control ◽  
Chain Reaction ◽  
Negative Results ◽  
Polymerase Chain ◽  
Positive Controls

To evaluate the sensitivity of polymerase chain reaction (PCR) to reveal known number of trypomastigote in the blood of mice, three separate experiments were done. First: To eight samples of 500mul of normal mice blood, one aliquot of 1, 2, 3, 4, 5, 10, and 50 trypomastigotes respectively, were added. Second and third: 10 aliquots with 1 and 10 with 2 trypomastigotes were added to samples of 500mul of normal mice blood. Positive control: 500mul of blood containing 100,000 trypomastigotes. For kDNA minicircles amplification by PCR the primers:S35 and S36 were used. PCR revealed products of 330 b.p in the positive controls. When only one sample with the aliquots of 1 or 2 trypomastigotes was examined, results were negative; results were positive with aliquots of 3 to 50 trypomastigotes. In the 2nd and 3rd experiments, 9/10 aliquots with one parasite and 9/10 with 2 trypomastigotes were positive revealing a high sensitivity of this reaction. In conclusion, the presence of one single parasite in 500mul of blood, is enough for a positive PCR. This method could be used as a complement to the various parasitological cure tests in treated mice, when low volumes of blood are individually examined.

Development of Unique Bacterial Strains for Use as Positive Controls in the Food Microbiology Testing Laboratory

2008 ◽  
Vol 71 (11) ◽  
pp. 2301-2306 ◽  
Author(s):  
BURTON W. BLAIS ◽  
AMALIA MARTINEZ-PEREZ ◽  
MARTINE GAUTHIER ◽  
RAYMOND ALLAIN ◽  
FRANCO PAGOTTO ◽  
...  
Keyword(s):  
Nalidixic Acid ◽  
Selective Pressure ◽  
Positive Control ◽  
Food Microbiology ◽  
Escherichia Coli O157 ◽  
Control Strain ◽  
Wild Type ◽  
Bacterial Strains ◽  
Mutant Strains ◽  
Positive Controls

Nalidixic acid–resistant (NalR) mutants of Salmonella enterica serovar Berta and Escherichia coli O157:H7 were derived from wild-type laboratory cultures to serve as distinguishable control strains for routine use in food microbiology testing programs. The prevalence of the NalR phenotype among different bacteria was verified using panels of related and unrelated strains with the ability to grow vigorously on plating media containing nalidixic acid, being restricted to the NalR mutants. The NalR phenotype was stable in both mutant strains over several generations in the absence of selective pressure and enabled their differentiation from wild-type bacteria on the basis of their ability to grow on plating media containing nalidixic acid. A similar approach for the development of a distinguishable Listeria monocytogenes control strain was not possible due to the inherent resistance of this organism to nalidixic acid. Instead, an L. monocytogenes isolate with rare genotypic and serologic features was identified as a possible candidate to serve as a unique and distinguishable positive control strain.

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