Preserved Blood Samples Research Articles

P-1192. Association between adeno-associated virus 2 and pediatric acute hepatitis of unknown etiology in Japanese children

Abstract Background Outbreaks of acute hepatitis of unknown etiology (AHUE) in children were reported in Western countries in 2022. Previous studies found that the adeno-associated virus 2 (AAV2) and other viruses, such as human adenovirus (HAdV) and human herpesvirus-6 (HHV-6), were frequently detected in patients with AHUE. As HAdV and HHV-6 are known as “helper viruses” that facilitate AAV2 replication, co-infection with AAV2 and helper viruses has been postulated as a potential trigger of AHUE. However, whether hepatitis associated with AAV2 existed prior to the AHUE outbreaks in 2022 has not yet been investigated. We aimed to investigate the association between AAV2 and pediatric acute hepatitis in Japanese children as well as the incidence of AAV2-related hepatitis before 2022. Visual abstract Methods Preserved blood samples from 49 pediatric patients (median age: 3 years) with acute hepatitis of unknown etiology between 2017 and 2023 were retrospectively analyzed. Blood samples from 50 children with acute illnesses, such as infectious diseases and 50 children with chronic conditions were used as controls. Viral DNA loads were quantified using real-time PCR. Results AAV2 DNA was detected in 12% (6/49) of acute hepatitis cases (median age: 2.5 years), but in only one acute illness case and none of the chronic condition control cases. The concentration of AAV2 DNA (range: 2.5×103 to 5.0×108 copies/mL) in the six patients in the acute hepatitis group was higher than that in the patient in the acute-illness control case (7.8×102 copies/mL). Co-infection with one or more helper viruses, including HAdV, HHV-6, cytomegalovirus, and Epstein–Barr virus, was observed in five of six AAV2-positive acute hepatitis cases. Conclusion Our results indicate the sporadic occurrence of severe pediatric hepatitis associated with AAV2 infection in Japan prior to AHUE outbreaks in Western countries in 2022. Conversely, the detection rate of AAV2 in this study was much lower than that of AHUE in Western countries, suggesting that the outbreak of AHUE related with AAV2 is an exceptional phenomenon associated with mitigation measures against COVID-19. Our findings suggest that coinfection with AAV2 and helper viruses plays a role in the development of severe hepatitis. Disclosures All Authors: No reported disclosures

Identification of 5-aminometonitazene and 5-acetamidometonitazene in a postmortem case: are nitro-nitazenes unstable?

In recent years, the use of 2-benzylbenzimidazole opioids ('nitazenes') has increased with them becoming one of the most prominent synthetic opioid subclasses of novel psychoactive substances. With the increased prevalence, there is also a concern of the dangers to public health with the use of nitazenes due to their high potency especially with polypharmacy. To aid in the detection of such compounds, it is important that forensic toxicology laboratories maintain up-to-date compound libraries for drug screening methods and that sensitive analytical instrumentation is available to detect the low blood/plasma concentrations of more potent drugs. This includes not only the compounds themselves but also potential metabolites and/or degradation products. Metonitazene is a 'nitro-nitazene' with a nitro group at position 5 of the benzimidazole ring. As a nitro-nitazene, there is a potential for bacterial degradation of metonitazene to 5-aminometonitazene, as occurs with nitro-benzodiazepines. In this study, we provide evidence from a postmortem (PM) case of degradation of metonitazene in unpreserved PM blood using liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ-MS), and putative identification of the degradation/metabolic products 5-aminometonitazene and 5-acetamidometonitazene by liquid chromatography-quadrupole time-of-flight mass spectrometry. The results from LC-QQQ-MS analysis indicated that there did not appear to be such degradation in preserved (fluoride/oxalate) blood. These results suggest that nitro-nitazenes may be subject to similar in vitro stability/degradation issues as nitro-benzodiazepines. These breakdown products should be added to instrument libraries to aid in the detection of the use of nitro-nitazenes, and nitro-nitazenes should be quantified in preserved blood samples where available.

Abstract 3705: Development and analytical validation of a novel assay for HER2 assessment on circulating tumor cells using Parsortix® isolation and BioView imaging technologies

Abstract Background. HER2 testing in tissue biopsies is extensively performed in breast cancer (BC) patients. However, tissue biopsy is invasive and repeated examination is impractical. Additionally, lack of concordance between primary tumor and metastatic sites are often observed. Liquid biopsy, on the other hand, enables less invasive and recurrent sample collection for ongoing monitoring. In this study, we developed an end-to-end Research Use Only assay for concomitant evaluation of HER2 gene amplification and protein overexpression in circulating tumor cells (CTCs). Methods. Blood was drawn from healthy volunteers into Streck Cell-Free DNA tubes, spiked with SKBR3 (HER2+) and Hs 578T (HER2-) breast cancer cells and processed on ANGLE's Parsortix® PC1 System, a microfluidic device that captures CTCs from metastatic breast cancer patients’ blood based on their size and deformability, without relying on specific markers. Captured cells were harvested and spun onto positively charged slides and stained with the Portrait HER2 immunofluorescence (IF) panel, consisting of a nuclear dye, epithelial and mesenchymal markers, leukocytes’ markers, and an antibody targeting HER2. Slides were then de-stained and subjected to HER2 FISH using a commercially available kit. All slides were imaged twice using BioView Allegro Plus, a platform equipped with artificial intelligence for automated imaging, CTC candidate identification and reporting. First, an automated scan after IF was performed to identify CTCs based on marker expression and morphology profiles. Second, an automated relocation of the CTCs based on IF results and reimaging using higher magnification to ensure precise analysis of the FISH signals in single cells. The workflow is being implemented in a cohort of BC patients. Results. Following IF, HER2 protein expression in SKBR3 cells was significantly higher (p<0.0001) than that of HER2-negative cells, with 100% analytical specificity and sensitivity. Following FISH, 100% of the cells were de-stained and presented sharp HER2/CEP17 foci, with 96% of the SKBR3 cells and only 4% of the Hs 578T cells displaying HER2 amplification. Additionally, in a small cohort of metastatic BC patients’, 100% of the identified CTCs are retained and evaluable from IF to FISH. Conclusions. An assay integrating HER2 identification by IF and FISH to characterize CTCs in metastatic BC patients was successfully developed. The assay enables the identification of both HER2 protein and gene copy information from each target cell with minimal cell loss, with the potential for minimally invasive patient monitoring during treatment and better classification of patients who may qualify and benefit from HER2-targeted therapies. An added value of the assay is the practicality of utilizing preserved blood samples, allowing streamline batch shipping of clinical samples. Citation Format: Mariacristina Ciccioli, Alex Young, Laila Vlietinck, Chassidy Johnson, Anne-Sophie Pailhes-Jimenez. Development and analytical validation of a novel assay for HER2 assessment on circulating tumor cells using Parsortix® isolation and BioView imaging technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3705.

Development of an analytical system for dried blood spots for forensic toxicology: a case study of five common drugs and poisons

Dried blood spot (DBS) technology is a simple and convenient method for collecting, transporting, and storing blood samples on filter paper, and has numerous applications in the clinical, research, and public health settings. This technique is gaining popularity in the field of forensic science because it facilitates the rapid analysis of prohibited drugs in blood samples and offers significant advantages in toxicology scenarios such as drinking-driving screening, drug abuse detection, and doping detection. However, the lack of a standardized system and the fact that its stability and reliability have not been thoroughly researched and demonstrated limit its application in judicial practice in China. DBS samples can be prepared, stored, and analyzed in various ways, all of which may significantly affect the results. In this study, we developed a method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) that focuses on the preparation, pretreatment, analysis, and storage of DBS samples. A thorough investigation was conducted to examine the optimal preparation conditions, including the blood spot matrix, drying technique, and preprocessing parameters, such as the solvent and extraction method. Moreover, the analytical conditions, such as the mobile phase system and elution gradient, were established to facilitate the quantitative detection of methamphetamine, lidocaine, ketamine, fentanyl, and diazepam in both DBS and whole-blood samples. The impact of storage conditions, such as the temperature, humidity, and sealing, on the analytical results of the DBS and whole-blood samples was also examined. The results showed a strong linear relationship for lidocaine and fentanyl within the range of 0.5-100 ng/mL. Similarly, methamphetamine, ketamine, and diazepam exhibited good linearity within the range of 2-100 ng/mL. The coefficients of determination (r2) ranged from 0.9983 to 0.9997, and the limits of detection ranged from 0.2 to 0.5 ng/mL, indicating a high degree of correlation and sensitivity. Stability tests demonstrated that the five target substances remained stable in the DBS for 60 days, with the measured contents deviating from the nominal values by 15%. Moreover, the measurement results of the DBS samples were highly similar to those of the whole-blood samples, with mean percentage differences of 4.44%, 3.50%, 7.66%, 5.10%, and 5.25% for fentanyl, diazepam, ketamine, lidocaine, and methamphetamine, respectively. Throughout the 60-day storage period, the maintenance of temperatures of -20 and 4 ℃, as well as sealing and dry storage, was not necessary. Room temperature was the most practical storage environment for the DBS samples. The results for each target showed very small concentration differences between the whole-blood and DBS samples, indicating that the DBS samples were suitable for drug and poison analysis in blood. Furthermore, the DBSs exhibited high quantitative consistency with the whole-blood samples, rendering them suitable matrices for preserving blood samples. Because DBS samples are easy to handle and store, they can realize the lightweight preservation of blood samples and provide a novel solution for the analysis and preservation of blood samples in public security practice. We recommend conducting comprehensive validations before utilizing DBS for analysis, particularly in terms of quantification, to ensure the judicial reliability of the results.

Prevalence of Leukocytozoon spp. in Rescued Wild Birds in Korea

Leukocytozoon spp. is a protozoan that causes infection in the blood, causing economic losses to the poultry industry. The aim of this study was to investigate the prevalence of leukocytozoonosis in wild birds rescued from January 2019 to December 2020. The medical records and the preserved residual blood samples of rescued and treated wild birds were analyzed. After DNA extraction from the preserved blood samples, real-time polymerase chain reaction (PCR) was used to test the extracted DNA. A total of 336 wild birds were tested in this study, of which 3.6% (6/336) were positive. Leukocytozoonosis was detected in four bird species, two of which were winter migratory birds and two were summer migratory birds in Korea. The results of this study suggest that wild birds visiting or staying in Korea may be infected with the Leukocytozoon spp., and there is a possibility that the pathogen may be transmitted to other domestic or wild bird species or shared with each other. It is necessary to conduct an extensive investigation focusing on important migratory bird habitats and to analyze the genetic relationships between domestic and wild bird-origin pathogens.

The Effect of Wall Type and Environmental Conditions toward Blood Type Identification Success Rate in Identifying Criminal Evident

Findings of blood at the crime scene (TKP) can provide important information in criminal cases such as homicide. Blood findings at crime scenes are usually blood spots or traces of blood that can be found on various substrates. This study aims to determine the type of wall substrate and environmental conditions that have the ability to preserve blood better, prove by success rate in identifying blood type in ABO system until 336 hour of exposure. In this study, blood samples were exposed to three variations of wall substrates, namely plastered walls, plastered and painted walls, plastered and oil-painted walls; and in two different environmental conditions (indoor and outdoor). The method used to identify blood type in this study was absorption elution. First, blood samples from substrate was transferred to a gauze by NaCl 0,98%. Then a confirmation test was carried out to ensure that the gauze sample contains a blood sample. Positive results of the confirmation test were indicated by a change in the color of the sample to bluish green when it is dripped with H2O2 and Leuco Malachite Green (LMG). The positive sample was then dripped with antiserum to determine the blood type. The success of identification of blood groups was indicated by the agglutination in the samples that were dripped with antisera A, because the blood samples used in this study are group A. The results showed that the success rate of blood group identification on the three types of substrates in the indoor environment for 336 hours was 100%. Meanwhile, in the outdoor environment, only blood samples exposed to plastered wall substrate and wall paint for 264 hours could be identified for their blood type. It can be concluded that the three types of wall substrates have the same ability to preserve blood samples for up to 336 hours of exposure, but the environmental conditions that give the best percentage of successful blood group identification were in indoor conditions. For further research is recommended to focus on external environmental factors that have the most influence on the success of blood group identification in dried blood samples.

Abstract 2223: High-purity isolation method of rare cells for molecular analyses using microfluidic chip type cell sorter

Abstract Background: Liquid biopsy using noninvasive sampling of blood is rapidly advancing in cancer diagnostics. Circulating tumor cells (CTCs) are intact tumor-derived cells and are potentially utilized for further molecular analyses. Capturing rare CTCs with high purity, however, remains technically challenging. Here we report the results of preclinical evaluation of the multistep sorting method using On-chip Sort system (On-Chip Biotechnologies) for high-purity capture and isolation of rare cells in the blood for further molecular analyses. Materials and Methods: Non-small-cell lung cancer cell lines PC-9, NCI-H1975 and A549 were used for this preclinical study. Cells were spiked into 4 mL of peripheral blood form healthy donors. After hemolysis and fixation, the samples were labeled with PE/Cy7-CD45 and FITC-cytokeratin (CK) antibodies and Hoechst for nuclei staining followed by three multiple sorting steps based on positive staining for CK and negative staining for CD45. For evaluating the potential of recovered tumor cells for genome sequencing, recovered NCI-H1975 cells, known to harbor EGFR L858R and T790M mutations, from spike-in samples were subjected to next-generation sequencing after whole genome amplification. To further establish the rare cells detection using the preserved blood samples, PC-9 cells were spiked the peripheral blood from healthy donors and transferred to the preservative tubes (Streck, Omaha, NE) and processed and sorted between 24 and 48 hours after the sample preparation. Results: Up to 50 PC-9 and A549 cells were spiked into 4 mL of peripheral blood and recovery rate of both cells were as high as 70% and recovered white blood cells were as low as 20% after three cycles of sorting steps, suggesting the sufficient recovery and purity of tumor cells for further analyses. One NCI-H1975 cells were also spiked into 4 mL of peripheral blood and recovered cells were subjected to next-generation sequencing after whole genome amplification. Both EGFR L858R and T790M mutations were successfully called and mutant allele frequencies of these gene mutations were as high as those called from the control DNA which were extracted from 1000 NCI-H1975 cells. PC-9 cells (0, 10, 100, 500 cells) were spiked into 20 mL of peripheral blood and split into two preservative tubes and the recovery rate ranged from 55.0 to 61.3%, suggesting the robustness of the multistep sorting method when preservative tubes were used. Good correlation was also observed between the observed number and the expected number of cancer cells demonstrating the linearity with a correlation coefficient (R2) of 0.99. Conclusions: We confirmed that rare cancer cells in the blood can be captured and isolated by the multistep sorting method using On-chip Sort system and can be utilized for further molecular analyses Results also suggest that this method allows us to deal with the preserved Citation Format: Yasuhiro Koh, Mio Ikeda, Shunsuke Teraoka, Masayuki Ishige, Yuu Fujimura, Kazuo Takeda, Nahomi Tokudome, Yuichi Ozawa, Hiroki Ueda, Nobuyuki Yamamoto. High-purity isolation method of rare cells for molecular analyses using microfluidic chip type cell sorter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2223.

Effect of blood storage conditions on the integrity of human DNA

A study was performed in blood samples, collected from 15 healthy individuals during July to December 2017, to evaluate the integrity of DNA by preserving blood samples at three temperatures i.e. 4, -20 and -80oC as against the fresh sample (control) for an incubation period of 30 days. DNA was extracted by standard protocol of phenol-chloroform and salting out method. The effect of temperature on DNA quality and quantity was analyzed spectrophotometerically, followed by amplification of Cytochrome p450 1A1 (CYP1A1) gene. In this prognosis, the study revealed that higher purity was attained at 4oC, yield and concentration at −20oC on extraction by phenol-chloroform. On contrary, in salting out method maximal yield and concentration was acquired at 4oC and purity at −20oC. Comparison of methods showed that phenol-chloroform method gave best result. DNA obtained from preserved blood samples is appropriate for amplification and further studies.

Optimal DNA extractions from blood on preservation paper limits conservation genomic but not conservation genetic applications

The acquisition and preservation of biological samples from rare and elusive wildlife species poses many challenges. Opportunities for collection are often limited and valuable DNA samples can degrade in unfavorable field conditions. Blood samples on preservation paper are commonly used across a wide range of taxa as a convenient and non-destructive method to obtain and store DNA. Preserved blood samples have been successfully used in a variety of genetic applications, primarily using traditional genetic markers and sequencing methods such as microsatellites and DNA barcoding. However, genomic methods are increasingly used for species of conservation concern. Genomic methods require large quantities of high-quality DNA and paper preservation methods can limit DNA availability, especially for mammals which have non-nucleated blood cells. Thus, high-yield DNA extraction methods are required to providing adequate unfragmented DNA for downstream genomic applications. Using a nested experimental design, we tested multiple DNA extraction methods and method modifications on mammalian blood stored on three common types of preservation paper. Whatman FTA classic cards with a modified extraction protocol using Qiagen QIAamp kits yielded the most DNA. We further tested the DNA extracted from FTA cards in microsatellite and genomic sequencing and found that FTA card samples were not adequate for genomic applications. For future mammalian blood collection efforts and genetic studies, researchers should weigh the need for high quantity, unfragmented DNA against the convenience of preservation paper. We recommend that researchers optimize their extraction methods and consider collecting fresh blood or tissue for genomic applications.

The sarcopenia index: A novel measure of muscle mass in lung transplant candidates.

Frailty, including low muscle mass, is an emerging risk factor for poor outcomes after lung transplant. The sarcopenia index (SI)-(serum creatinine value/cystatin C value)×100-is a novel blood test to approximate muscle mass. We sought to validate SI among lung transplant patients. We retrospectively identified adult lung transplant recipients from 2000 through 2012 at our institution who underwent computed tomography within 1year before transplant and had preserved blood samples. Creatinine and cystatin C values were measured using the samples and used to calculate SI. Muscle mass was estimated by computed tomographic measurement of skeletal muscle cross-sectional surface area (SA) at the L1 to L3 vertebral levels. Correlation between SI and SA was evaluated. Of 28 patients meeting eligibility criteria, most were white (96%) and men (54%). Median (interquartile range) body mass index, SI, and SA were 25.9 (22-30) kg/m2 , 106 (91-119), and 157 (113-195) cm2, respectively. The Pearson correlation coefficient between SI and SA was significant at L2 (0.43; P=.02) and L3 (0.41; P=.03). Sarcopenia index is a potentially objective measure for estimating muscle mass that is noninvasive and less expensive. Sarcopenia index could be considered in lung transplant candidate selection following prospective validation in larger cohorts.

2% of Healthy Newborns Reveal ETV6-RUNX1 Fusion By Genomic Inverse PCR for Exploration of Ligated Breakpoints (GIPFEL)

Pediatric acute lymphoblastic leukemia (ALL) is characterized by preleukemic recurrent chromosomal translocations that emerge in utero. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell precursor patients. A previous study suggested that ETV6-RUNX1-positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1-positive ALL in children (1/10,000) by a factor of 100. This finding strongly indicated that secondary cooperating oncogenic hits were necessary for development of overt leukemia. However, later studies could not confirm this high frequency.To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR-based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based retrospective screening of 300 cord blood samples from Danish newborns.The GIPFEL method is capable of detecting the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In contrast to previously used RNA-based methods, it relies on DNA as sample material, which is more stable than RNA. In the case of ETV6-RUNX1-positive leukemia GIPFEL exploits the unique presence of a genomic fragment joining material from chromosome 12 and 21. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. The ligation site is independent of the translocation point within the individual DNA circle. Digestion of the breakpoint regions of the ETV6 and RUNX1 gene with the restriction enzyme SacI generates fragments smaller than 50 kb. Primer pairs amplify the complete set of theoretically predicted circularized fragments requiring 37 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from mononuclear cells from cord blood samples of 300 newborns that were cryopreserved within 24 h (median 12 h) from birth. After B cell enrichment and column purification of DNA, the DNA was subjected to SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. Samples that screened positive underwent one further demultiplexed PCR, agarose gelelectrophoresis and Sanger sequencing to validate the result and to identify the breakpoint region. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product. In previously published proof-of-principle blinded studies we tested 61 samples obtained from ETV6-RUNX1-positive ALL patients. Without any unspecific result, 64% for ETV6-RUNX1 fusion genes were detected in that sample set. The sensitivity of the technique was estimated to be 10-4, i.e. one translocation carrying cell within 10,000 normal cells can theoretically be detected.Within the analyzed cohort of 300 healthy newborns 6 screened positive for the ETV6-RUNX1 translocation (2%) (Table 1). Further 700 cord blood samples are currently screened.Table 1: 6 of 300 cord blood samples from healthy newborns screened positive for the ETV6-RUNX1 translocation using the GIPFEL technique (Fueller E*, Schaefer D* et al. PloS One 2014, 9(8): e104419). Number of the positively tested healthy newborn within the cohort, used primers, and introns of RUNX1 and ETV6 affected by the translocation are presented.Our results indicate that the actual incidence of ETV6-RUNX1-positive cells in healthy newborns might be even higher than previously assumed, potentially due to instability of the ETV6-RUNX1 RNA transcript in preserved cord blood samples. This would hint at a comparably low penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and further strengthen the importance of secondary environmentally caused or spontaneously occurring cooperating oncogenic lesions for ETV6-RUNX1-positive childhood leukemia to emerge. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 401: Preservation of cells in blood and culture

Abstract Introduction: Liquid biopsies and cell-based therapies are emerging approaches for the treatment of cancer. Accuracy of diagnosis using liquid biopsies and effectiveness of treatment using cell-based therapies will rely upon the preservation cells for both viability and the information contained in them. Experimental procedures: Biomatrica's proprietary chemical library was used to develop formulations that could preserve cells in multiple formats. Cell preservation was assessed by a variety of cell-based and molecular techniques such as microscopy, flow cytometry and real-time PCR. Results: Blood samples spiked with circulating tumor cells (CTCs) were preserved for several days at room temperature to allow for downstream morphological and transcriptomic analyses. CTC recoveries in preserved samples were as much as 4 times those in non-protected samples over 4 days. Changes in RNA expression of CTC-specific genes in preserved samples ranged from 0 to +/- 2 delta Ct values, while in non-protected samples, changes ranged from 0 to +/-5 delta Ct values over 4 days. In some formulations, CTC viabilities were as much as twice the viabilities of CTCs in non-protected samples. Supporting these viability results was a demonstrated culturability of isolated CTCs. Furthermore, cell-free DNA in plasma of preserved blood samples changed by less than 2 Ct values over 4 days, whereas in control samples, plasma DNA changed by as much as 10 Ct values. Hemolysis in preserved blood samples was also lower by as much as 69% compared to non-protected samples. In a separate set of experiments, activated T-cells were stored in the presence of common buffers (RPMI, PBS) or formulations at room temperature. T-cells cultured with formulations displayed greater 40 to 60% greater cell viability after 3 days compared to those stored in common buffers. Conclusions: Biomatrica has developed a library of cell preservatives that can be used to preserve cells in blood samples and in cell culture. The ability to recover cells in blood samples and determine their genetic information would enable monitoring of a patient's cancer status and personalizing drug treatments. In addition, cell preservation may allow further advances in cell-based therapies as cells could be more reliably recovered from patients, modified (e.g. CAR-T, stem cells), and administered to patients. Citation Format: Cecille Browne, Dan Lu, Daniela Roth, Vasco Liberal. Preservation of cells in blood and culture. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 401.

Stability of Water Soluble Antioxidants in Cattle Blood Collected in Different Media

The study was carried out to compare between different media used to preserve blood samples taken in the field, and to determine in which of these media is the water soluble antioxidative capacity (ACW) is most stable, because sometimes it takes long time for the blood samples taken in the field to be transported to the laboratory where different biochemical tests are made, in this time during transport the samples underly many factors that have an effect on the content of the samples under study like light, temperature, and time itself. Also the effect of centrifugation was tested to see weather direct or delayed centrifugation has an effect on the water soluble antioxidative capacity of the blood.There are no studies related directly to choosing the proper medium for preserving blood samples when the water soluble antioxidative capacity will be tested. In this study the highest values were achieved using serum samples, and this was the reason why serum tubes were chosen in the rest of the study.In this study four different media were tested, and compared with each other, the measured ACW values in the different media were arranged as follows:serum>NaF-LiH>LiH> EDTA.DOI: http://dx.doi.org/10.3329/bjvm.v12i1.20461 Bangl. J. Vet. Med. (2014). 12 (1): 35-40

Anti-Saccharomyces cerevisiae Autoantibodies in Autoimmune Diseases: from Bread Baking to Autoimmunity

Saccharomyces cerevisiae is best known as the baker's and brewer's yeast, but its residual traces are also frequent excipients in some vaccines. Although anti-S. cerevisiae autoantibodies (ASCAs) are considered specific for Crohn's disease, a growing number of studies have detected high levels of ASCAs in patients affected with autoimmune diseases as compared with healthy controls, including antiphospholipid syndrome, systemic lupus erythematosus, type 1 diabetes mellitus, and rheumatoid arthritis. Commensal microorganisms such as Saccharomyces are required for nutrition, proper development of Peyer's aggregated lymphoid tissue, and tissue healing. However, even the commensal nonclassically pathogenic microbiota can trigger autoimmunity when fine regulation of immune tolerance does not work properly. For our purposes, the protein database of the National Center for Biotechnology Information (NCBI) was consulted, comparing Saccharomyces mannan to several molecules with a pathogenetic role in autoimmune diseases. Thanks to the NCBI bioinformation technology tool, several overlaps in molecular structures (50-100 %) were identified when yeast mannan, and the most common autoantigens were compared. The autoantigen U2 snRNP B″ was found to conserve a superfamily protein domain that shares 83 % of the S. cerevisiae mannan sequence. Furthermore, ASCAs may be present years before the diagnosis of some associated autoimmune diseases as they were retrospectively found in the preserved blood samples of soldiers who became affected by Crohn's disease years later. Our results strongly suggest that ASCAs' role in clinical practice should be better addressed in order to evaluate their predictive or prognostic relevance.

The effect of Point Mutations in Dihydrofolate reductase genes and Multidrug resistance gene 1-86 on treatment of falciparum malaria in Sudan

One of the major problems to the treatment of malaria is the emergence and spread of parasite resistant to antimalarial drugs. Due to increased chloroquine (CQ) resistance, the antifolate combinations are becoming important in the chemotherapy of falciparum malaria. However, resistance to antifolate exists and they are still effective in the above combinations. This study aimed at determining the prevalence of antimalarial drug resistance markers in P. falciparum isolates, involving the detection of mutations at the mdr 1- 86 which associates with amodiaquine resistance, and dhfr mutations associated with SP resistances. The dot-blot/ probe hybridization, which is more sensitive and specific; it detects parasitaemia of less than 100 parasites/microl of blood, and can identify a minority parasite genotype down to 1% in a mixture, was adopted to determine multi-drug resistance (mdr1-86) to show the correlation of Amodiaquine (AQ) resistance and PCR/ RFLP adopted to determine dihydrofolate reductase (dhfr) baseline resistance to Sulphadoxine- Pyrimethamine (SP) resistance in Nubian region of southern Sudan. A randomized open label trial of Artesunate (AS) + SP and AS+ SP was carried out in children less than 5 years. Molecular analysis of filter paper preserved blood samples collected was carried out to provide a baseline estimate of allele prevalences. Baseline of the allele prevalence of the mdr1 86 locus in the AS+ AQ was successful for 80 isolates: 71(8.11%) carried parasites harbouring the mdr1-86 Tyr resistance allele, while 7 (89.19%) carried mdr1-86 Asn sensitivity allele and 2 (2.7%) were of mixed infection, having both resistance and wild type allele. Overall, the prevalence of the dhfr point mutation, codon 51, 59 and 108: 82.5% (132/160) carried mutations at dhfr (N51I, C59R or S108N), but triple mutants were rare (3.1%) in the AS + SP arm. The research provides the evidence that mutations present in dhfr and mdr1 86 has a significant effect on the type of treatment following SP and AQ chemotherapy. SP resistance may spread rapidly, and AS + AQ is likely to be a better option, provided AQ use is restricted to the combination. The significance of the study shows that definitely combination of drugs improves SP therapy at the study site.

A novel method for the diagnosis of drowning by detection of Aeromonas sobria with PCR method

The acid digestion method has been widely used for the diagnosis of death by drowning, but it is not always sensitive. However, there has been no definitive method to replace acid digestion until now. We speculate that bacteria are more useful markers than plankton for the diagnosis of death by drowning. In this study, from the preserved blood samples of 32 freshwater drowning cases, specific DNA fragments of Aeromonas sobria, one of the most common aquatic bacteria, were examined using PCR. The DNA fragments of the bacterium were detected from 27 of 32 cases with first round PCR or nested-PCR. The remaining 5 cases in which bacterial DNA was not detected had longer storage periods for the blood samples and shorter time intervals from drowning to death. These results indicate that the present method can be applied to the diagnosis of death by drowning.

Establishment of experimental conditions for preserving samples of fish blood for analysis with both comet assay and flow cytometry

When environmental analysis is performed, the high number of samples required and handling conditions during the transport of these samples to the laboratory are common problems. The comet assay is a useful, highly sensitive tool in biomonitoring. Some studies in the literature aim to preserve slides in lysis solution for use in the comet assay. Until now, however, no efficient methodology for preserving blood samples for this assay has been described. Because of this, the present report aimed to establish the proper conditions for samples maintenance prior to comet assay analysis. Samples were conserved in three different solutions: a high protein concentration solution (fetal bovine serum—FBS), an anticoagulant agent (a calcium chelator – ethylenediaminetetracetic acid – EDTA), and a salt buffered solution (phosphate buffered saline—PBS). Therefore, peripheral blood samples of Rhamdia quelen specimens were collected and maintained in these solutions until testing at 72 h. Analyses of DNA fragmentation via the comet assay and cell viability via flow cytometry were performed at intervals of 24 h. The results showed that samples maintained in FBS were preserved better; this was followed by those preserved in PBS and then last by those preserved in EDTA. In conclusion, blood samples from freshwater fish can be preserved up to 48 h in fetal bovine serum at 4 °C in the absence of light. In this period, no DNA fragmentation occurs. We thus describe an excellent method of sample conservation for subsequent analysis in the laboratory.

Limitations in the use of potassium dichromate as a blood preservative for the analysis of organohalogenated compounds: Two month results

For analysis of organochlorine contaminants in human tissue, the “gold standard” for preservation, storage, and shipping is usually freezing. However, this method can be difficult, if samples are taken in remote areas, and costly, when the samples must be shipped on dry ice. Therefore, a more simple and cost effective method of preservation is essential for remote field work. Potassium dichromate (K 2Cr 2O 7) has been successfully employed in the preservation of human and cows’ milk as well as chicken eggs. Our previous studies described the use of potassium dichromate for preservation of whole blood for analysis of dioxins, dibenzofurans, and PCBs. Potassium dichromate was found to successfully preserve blood at room temperature for 34 d with no significant differences in the measured concentrations of chemical contaminants or blood lipid level when compared to frozen samples. However, in a follow-up study, 3 months and 6 months of potassium dichromate preservation proved inadequate to preserve the samples for organic pollutant analysis. We noted that the lipid portion of the blood in the chemically preserved samples was declining in level or degrading, while the persistent organic pollutants remained intact at the same levels on a whole weight basis. To narrow down the window of efficacy for the use of potassium dichromate to preserve blood samples for analysis, the present study compared chemical preservation to freezing for an intermediate time period, 2 months. Similar to our previous findings at 3 and 6 months, at 2 months significant lipid degradation was observed in the chemically preserved samples. Chemically preserved samples had significantly higher levels of organochlorine contaminants (dioxins, dibenzofurans, and PCBs) when measured on a blood lipid basis but not on a wet weight basis compared to frozen samples. While 2 months of potassium dichromate preservation was not useful for obtaining accurate measure of dioxins, furans, and PCBs on a lipid basis, previous studies found this method of preservation to be useful for at least one month (Schecter, A., Pavuk, M., Päpke, O., Malisch, R., 2004. The use of potassium dichromate and ethyl alcohol as blood preservatives for analysis of organochlorine contaminants. Chemosphere 57, 1–7). However blood stored at −70 °C and at 22 °C with potassium dichromate gave similar results when expressed on a wet weight basis.

Evaluation of blood preservation methods in the performance of the WHO in vitro micro-test for Plasmodium falciparum in the field

In vitro drug susceptibility testing of Plasmodium falciparum must be conducted immediately after collecting a sample of the patient‘s blood; otherwise the parasites may weaken and the culture fail. Collecting blood samples from individuals in areas far from the field station or clinic where in vitro testing is conducted requires a reliable method of sample preservation during transportation. We examined and compared three different methods used to preserve blood samples in endemic areas in the Philippines. The three methods are as follows: the on-site method (test is conducted soon after blood sampling), flask culture method (sample is taken to the laboratory in a culture flask with medium) and EDTA tube method (sample is taken to the laboratory in a blood collection tube). The WHO in vitro micro-test for susceptibility of P. falciparum to chloroquine was performed using an AnaeroPack® system and a portable thermostat incubator. Evaluation of the three methods was based on schizont maturation, ease of handling, and risk of contamination during the test. The on-site and flask culture methods, but not the EDTA tube method, were effective for keeping the parasites viable. Furthermore, schizont maturation appeared better with the flask method than with the on-site method, especially in the control wells (drug-free wells). In addition, it was easier to perform the flask method than the on-site method. No contamination was observed using any of the methods. The results of the study suggest that the flask culture method is the most effective and useful way to preserve blood samples for the in vitro test and, moreover, that it aids in providing detailed field evidence of drug-resistant malaria.

Ether: stability in preserved blood samples and a case of ether-assisted suicide.

Ethyl ether was detected in the blood of a deceased individual who had inhaled it from a mask while hanging himself. This case led to an investigation into the stability of ether in stored blood samples as described herein. Ether was spiked, at three concentrations, into batches of porcine blood. The blood was apportioned into grey-stoppered collection tubes and stored refrigerated until analysis. The concentration of ether in the blood was assessed at intervals over a two-month period. Analysis was performed by headspace gas chromatography. This study has shown, for a concentration range of 0.1 to 5.0 mg/mL, ether is stable in preserved, refrigerated blood for at least two months.