Frozen Blood Samples Research Articles

Abstract 4148070: Clonal Hematopoeisis of Indeterminate Potential and Risk of Autopsy-defined Sudden Cardiac Death

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) has been associated with an increased risk of coronary artery disease (CAD) and cardiovascular (CV) mortality. Whether CHIP may affect myocardium beyond promoting CAD, or its potential association with sudden cardiac death (SCD), the most feared manifestation of CV disease, is unknown. Research Questions/Hypothesis: We hypothesize that CHIP-mutant macrophages infiltrate the myocardium and are associated with autopsy-defined arrhythmic death. Goals/Aims: (1) determine rate of CHIP at time of death in an unselected, countywide study of all incident sudden deaths; (2) evaluate whether CHIP-mutant macrophages infiltrate into myocardium; and (3) evaluate potential association of CHIP with autopsy-confirmed arrhythmic causes among countywide sudden deaths Methods/Approach: We used autopsy to adjudicate arrhythmic from non-arrhythmic (PE, stroke, overdose, tamponade) causes in 1,148 sudden deaths and 141 trauma control deaths in San Francisco County from 2011-23. DNA was extracted from frozen blood samples collected at time of death from 399 consented cases. Sequencing of 22 CHIP-associated genes was performed to ~2000x mean target coverage. All pathogenic/likely pathogenic CHIP variants at >2% variant allele frequency (VAF) were considered CHIP+. DNA was then isolated from left ventricular (LV) tissue sampled at autopsy in all CHIP+ blood cases and sequenced as above. Results: Of 399 consented cases, 70 blood samples (17.5%) were CHIP+ (range 2%-39.6%; mean age 70.6 years vs. 58.1 years for CHIP-). The most frequent mutant genes were DNMT3A, TET2, and ASXL1. Proportion of arrhythmic causes among sudden deaths was similar for CHIP+ vs. CHIP- cases (36 of 70 [52%] vs. 171 of 329 [51%]). Sequencing of available LV tissue from 61 CHIP+ blood cases revealed 67% (n=41) harbored the same CHIP mutation identified in blood at >0.2% VAF (range 0.2%-4.3%). Proportion of arrhythmic causes among sudden deaths was significantly higher in cases where both blood and LV were CHIP+ (24 of 41 [58.5%] vs. 5 of 19 [26.3%] CHIP+ blood/CHIP- LV, p=0.02). Conclusions: Prevalence of CHIP positivity at sudden death is similar to published studies and correlated with age. CHIP+ status in myocardial tissue but not peripheral blood was associated with arrhythmic causes of sudden death, suggesting a direct tissue effect of CHIP-mutant macrophages.

B-184 Purifying High-Quality Genomic DNA From Frozen Blood Samples Stored up to 1.5 Years at -20°C

Abstract Background Maintaining DNA integrity in blood samples, from transport to storage to processing, is crucial to the success of downstream applications, especially regarding diagnostic applications. Additionally, due to the invasive nature of collecting blood samples, minimizing resampling to avoid excess patient pain and cost of collection and transportation are important to consider. Having the option to store and maintain portions of blood samples in the freezer enables future retesting without recollection. Since -20°C freezers generally are more affordable, require less energy, and have smaller footprints than -80°C freezers, demonstrating frozen blood stability at -20°C temperatures could decrease sampling burdens for labs with facility constraints. Methods Whole blood was collected into 9mL vacuum tubes (HemaSure-OMXP) from healthy donors. Aliquots of whole blood and buffy coat samples were stored at -20°C and thawed for genomic DNA extractions (QIAamp DNA Blood Mini Kit, Qiagen) at a range of different timepoints; frozen whole blood samples were processed at 0, 49, 141, and 148 days and frozen buffy coat samples were processed at 7, 27, 36, 185, 246, 373, 394, 584, and 604 days. Purified gDNA was quantified with Qubit (Broad Range dsDNA Assay, ThermoFisher) and qualified with Nanodrop One Microvolume UV-Vis Spectrophotometer (ThermoFisher) and TapeStation (Genomic DNA ScreenTape, Agilent Technologies). Results No significant decrease was observed for DNA yield and quality between fresh and frozen whole blood samples. Average DNA yields from 200 μL whole blood were 4.5 ± 2.2 μg and 5.3 ± 1.2 μg for fresh and frozen blood respectively. Average DNA integrity (DIN) was 6.9 ± 0.4 and 7.1 ± 0.3 for fresh and frozen blood respectively. Donor differences in blood samples can help explain the variance in DNA yields reported. No significant decrease was observed for DNA yield and quality in buffy coat samples frozen for longer at -20°C. In total, 84 whole blood and 25 buffy coat samples were processed in this study. Conclusions Although buffy coat samples tended to have higher yields than whole blood, due to the higher amount of white blood cells present, both sample types provided sufficient amounts of gDNA for downstream applications. Freezing blood samples for future testing can be effective at reducing the need for resampling without sacrificing the nucleic acid yield or quality of fresh samples. Additionally, as technological advancements are made for equipment and assays, frozen blood samples can be retested and compared to previous results for improved diagnostic decisions, done without requiring recollection from labs and patients.

Association of Endothelial Cell Activation with Acute Kidney Injury during Coronary Angiography and the Influence of Recombinant Human C1 Inhibitor-A Secondary Analysis of a Randomized, Placebo-Controlled, Double-Blind Trial.

Acute kidney injury (AKI) as a result of iodinated contrast media (CM) has been linked to CM-induced renal ischemia and toxic effects on endothelial cells (EC). The recombinant human C1 inhibitor (rhC1INH) has been shown to influence EC activation. Secondary analysis of 74/77 (96%) participants of a double-blind, randomized, and placebo-controlled study that assessed the effect of rhC1INH on AKI. E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), and CC-chemokin-ligand-5 (CCL5) were determined in frozen blood samples over 48 h and analyzed according to the treatment group and renal outcomes. The mean age was 76.7 years, and 37 patients each received rhC1INH and placebo, respectively. In the entire study population, minor differences in median EC activation markers/CCL5 concentrations during the first 48 h compared to baseline were observed (e.g., E-selectin 27.5 ng/mL at baseline vs. 29.7 ng/mL on day 1, CCL5: 17.7 ng/mL at baseline vs. 32.2 ng/mL on day 2). Absolute changes in ICAM-1/E-selectin concentrations correlated with a higher peak change in urinary NGAL concentrations. However, AKI was not associated with significant changes in EC markers/CCL5. Last, no significant differences in serum concentrations of EC activation markers/CCL5 were evident between the placebo and the rhC1INH group. CM administration during coronary angiography only mildly activated ECs within the first 48 h, which does not explain subsequent AKI. The administration of rhC1INH was not associated with a reduction of EC activation or CCL5.

Establishing a method for the cryopreservation of viable peripheral blood mononuclear cells in the International Space Station

The analysis of cells frozen within the International Space Station (ISS) will provide crucial insights into the impact of the space environment on cellular functions and properties. The objective of this study was to develop a method for cryopreserving blood cells under the specific constraints of the ISS. In a ground experiment, mouse blood was directly mixed with a cryoprotectant and gradually frozen at −80 °C. Thawing the frozen blood sample resulted in the successful recovery of viable mononuclear cells when using a mixed solution of dimethylsulfoxide and hydroxyethyl starch as a cryoprotectant. In addition, we developed new freezing cases to minimize storage space utilization within the ISS freezer. Finally, we confirmed the recovery of major mononuclear immune cell subsets from the cryopreserved blood cells through a high dimensional analysis of flow cytometric data using 13 cell surface markers. Consequently, this ground study lays the foundation for the cryopreservation of viable blood cells on the ISS, enabling their analysis upon return to Earth. The application of this method in ISS studies will contribute to understanding the impact of space environments on human cells. Moreover, this method may find application in the cryopreservation of blood cells in situations where research facilities are inadequate.

Comparison of DNA damage in fresh and frozen blood samples: implications for the comet assay in human biomonitoring studies.

The use of the comet assay in large biomonitoring studies may present logistical and technical challenges because of the processing of numerous samples. Proper sample preservation becomes imperative to prevent spurious DNA breakage. Previous research has shown the feasibility of conducting the comet assay on frozen blood samples, highlighting the potential of freezing at - 80°C in preserving DNA integrity. Nonetheless, this approach presents challenges, including potential DNA damage during freezing and thawing, variability in processing, and the need for standardized protocols. Our objective was to evaluate whether there are comparable results in DNA migration assessed by the comet assay between fresh and frozen blood samples on a larger scale (N = 373). In our findings, elevated DNA migration was evident in frozen samples relative to fresh ones. Additionally, smoking, alcohol consumption, and season were linked to increased DNA damage levels in whole blood cells. Based on our results and available literature, conducting the comet assay on frozen blood samples emerges as a practical and efficient approach for biomonitoring and epidemiological research. This method enables the assessment of DNA damage in large populations over time, with samples, if properly cryopreserved, that may be used for years, possibly even decades. These observations hold significant implications for large-scale human biomonitoring and long-term epidemiological studies, particularly when samples are collected during fieldwork or obtained from biobanks. Continued method optimization and validation efforts are essential to enhance the utility of this approach in environmental and occupational health studies, emphasizing caution when comparing data obtained between fresh and frozen blood samples.

The Efficiency of the Alu Insertion Sequence in Discrimination Among some Individuals

Background: The Alu element is a widely distributed short interspersed nuclear element (SINE) in the human genome and has important applications in forensic science. The current study focused on assessing the effectiveness of Alu insertion polymorphism in forensic DNA profiling to identifying samples of some individuals living in the Babylon Governorate. Materials and methods: DNA was extracted From frozen blood samples (60) individuals were collected from Babylon - Al-Hilla Governorate from 8/8/2022 until 8/9/2022. Results: purified then a PCR technique Alu insertions were A(2q21.1)(111-115bp) 0.75, Alu deletion (0.25), B(8q23.1) (0.375) (0.625) C(13q34) (0.5)(0.5), D(15q23) (0.775) (0.225), E(16q23.3) (0.692) (0.308) and F(19q13.12)(0.616)(0.384) respectively.The alleles with Alu insertions at (2q21.1) were the most prevalent, whereas the Alu insertions at (8q23.1) were the least common. The similarity coefficient among individuals varied from 0.4 to 1, based on the proportion of genetic relatedness between them. Conclusions: Alu elements have efficacy in families’ discrimination it can be used more than one site, set of Alu insertion sequences should be used to accurate results.

Assessing the impact of long-term storage on the quality and integrity of biological specimens in a reproductive biobank.

Biobanks hold a pivotal role in facilitating translational and clinical research endeavors. However, the effects of prolonged storage on frozen blood samples analytes are not well defined yet. The aim of this study was to investigate the long-term stability of the quality of DNA, RNA, and endocrine markers within blood samples amassed from the biobank over the past 11 years. The results show that the overall quality and integrity of DNA remained not significantly influenced. However, RNA integrity and purity displayed substantial deterioration as storage duration increased, to ensure high-quality RNA for downstream analyses, advised to prioritize using blood samples stored within 3 years. Furthermore, the study examined the influence of storage time on endocrine markers. Through repeated measures ANOVA and linear regression analyses, it was evident that storage duration significantly influenced the levels of endocrine markers. This insight aids researchers in selecting appropriate markers for their investigations and augments the precision and dependability of results when dealing with long-term stored samples.

Consistency of metabolite associations with measured glomerular filtration rate in children and adults.

There is interest in identifying novel filtration markers that lead to more accurate GFR estimates than current markers (creatinine and cystatin C) and are more consistent across demographic groups. We hypothesize that large-scale metabolomics can identify serum metabolites that are strongly influenced by glomerular filtration rate (GFR) and are more consistent across demographic variables than creatinine, which would be promising filtration markers for future investigation. We evaluated the consistency of associations between measured GFR (mGFR) and 887 common, known metabolites quantified by an untargeted chromatography- and spectroscopy-based metabolomics platform (Metabolon) performed on frozen blood samples from 580 participants in Chronic Kidney Disease in Children (CKiD), 674 participants in Modification of Diet in Renal Disease (MDRD) Study and 962 participants in African American Study of Kidney Disease and Hypertension (AASK). We evaluated metabolite-mGFR correlation association with metabolite class, molecular weight, assay platform and measurement coefficient of variation (CV). Among metabolites with strong negative correlations with mGFR (r < -0.5), we assessed additional variation by age (height in children), sex, race and body mass index (BMI). A total of 561 metabolites (63%) were negatively correlated with mGFR. Correlations with mGFR were highly consistent across study, sex, race and BMI categories (correlation of metabolite-mGFR correlations between 0.88 and 0.95). Amino acids, carbohydrates and nucleotides were more often negatively correlated with mGFR compared with lipids, but there was no association with metabolite molecular weight, liquid chromatography/mass spectrometry platform and measurement CV. Among 114 metabolites with strong negative associations with mGFR (r < -0.5), 27 were consistently not associated with age (height in children), sex or race. The majority of metabolite-mGFR correlations were negative and consistent across sex, race, BMI and study. Metabolites with consistent strong negative correlations with mGFR and non-association with demographic variables may represent candidate markers to improve estimation of GFR.

The Association between Single Nucleotide Polymorphisms rs1042522 and rs1642785 in the TP53 gene and Acute Myeloid leukemia in a sample of the Baghdad/ Iraq population

يمثل سرطان الدم النخاعي الحاد (AML) أكثر أنواع سرطان الدم الحاد انتشارًا بين البالغين تصل نسبته 80% من جميع الحالات. الجين المثبط للورم TP53 هو جين تمت دراسته بشكل متكرر في مرض السرطان .والطفرات في هذا الجين تسبب ما يقارب 50% من الامراض السرطانية. هدفت هذه الدراسة إلى تقييم العلاقة بين اثنين من تعدد أشكال النوكليوتيدات المفردة في جين TP53: rs1042522 ، rs1642785 ومجموعة من المرضى العراقيين الذين يعانون من التشخيص الاولي لسرطان الدم النخاعي الحاد. جمعت عينات الدم من ستين مريضاً 26 ذكور و 34 أناث وستين عينة سيطرة 26 ذكور و 34 أناث متوافقين في الجنس والعمر والعرق. تم استخلاص الحمض النووي الجيني من عينات الدم المجمدة بالكامل باستخدامEasy Pure® Blood Genomic Kit . ثم تم قياس النقاوة والتركيز باستخدام مقياس الطيف الضوئي Nano drop NAS-99. حيث تراوحت قراءات التركيز بين (7-55 نانوغرام / ميكرولتر) وتراوحت النقاوة بين 1.78 - 1.9 تم استخدام تقنيةreal time PCR (High resolution melt (HRM للكشف عن تعدد اشكال النيوكليوتيدات المفردة و كانت تكرارات النمط الجيني لجين TP53 متوافقة مع توازن هاردي-واينبرغ مع وجود فروق معنوية ذات دلالة إحصائية p≤0.05 بين الأنماط الجينية لمجموعة السيطرة والمرضى. كان تكرار النمط الجيني rs1042522 مختلفًا بشكل كبير بين المرضى وافراد السيطرة p = 0.0001، وكان المشاركون الذين لديهم النمط الوراثي GA أكثر عرضة للإصابة بسرطان الدم النخاعي الحاد OR=7.8, 95% CI 3.2–18.4, p = 0.0001. بالإضافة الى تكرار النمط الجيني rs1642785 مختلفًا بشكل كبير بين المرضى و افراد السيطرة p=0.002 والذين يحملون النمط الجيني GA أكثر عرضة للإصابة بسرطان الدم النخاعي الحاد OR=3.5, 95% CI 1.5–8.12, p=0.002.

The Micronucleus Assay on Cryopreserved Whole Blood.

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, and in vitro radiosensitivity testing. This cytogenetic method is based on the detection of micronuclei in binucleated cells resulting from chromosomal fragments lagging during cell division. Fresh whole blood samples are the most preferred sample type for the CBMN assay. However, the disadvantages of working with fresh blood samples include immediate processing after blood collection and the limited number of repeated analyses that can be performed without extra blood sampling. As the need for fresh blood samples can be logistically challenging, CBMN assay on cryopreserved whole blood samples would be of great advantage, especially in large-scale patient studies. This paper describes a protocol to freeze whole blood samples and to perform the CBMN assay on these frozen blood samples. Blood samples from healthy volunteers have been frozen and thawed at different time points and then, subjected to a modified micronucleus assay protocol. The results demonstrate that this optimized procedure allows the performance of the CBMN assay on frozen blood samples. The described cryopreservation protocol may also be very useful for other cytogenetic assays and a variety of functional assays requiring proliferating lymphocytes.

Determinants of Response to Anti CD-19 CAR-T Cells for Diffuse Large B-Cell Lymphoma in Pre-Treatment Peripheral Blood Mononuclear Cells Using Single-Cell RNA-Seq

Background: Chimeric antigen receptor T (CAR-T) cell therapy has become the standard of care in patients with relapsed/refractory diffuse large cell lymphoma (R/R DLBCL), providing durable remission in 40% of patients. Lack of response is likely to be attributed to inter- and intra-patient variability, including PBMC heterogeneity and apheresis material composition, CAR-T infusion product, in-vivo expansion and activity of CAR-T cells and the development of tumor-intrinsic mechanisms. Current methods for evaluating CAR-T cell activation and potency have limited predictive value for clinical outcomes. To address this challenge, we performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) obtained from patients with R/R LBCL patients at the time of apheresis for the manufacturing of anti-CD19 CAR-T cell therapy. Methods: Between 10/2020 and 03/2022, 31 patients who underwent lymphopheresis were enrolled. Samples were obtained - 1) At day of lymphopheresis from peripheral blood; 2) On the day of infusion of CAR-T from the product; and 3) On day +7 after infusion from peripheral blood. All patients had day +30 PET-CT. In addition, PBMCs samples from healthy donors were used as control to analyze baseline characteristics compared to the DLBCL patients. PBMCs from patients of healthy donors were purified from frozen blood samples by density gradient separation, stained using human CD45 antibody and loaded onto a 10x Chromium system. libraries were generated using the 10X Genomics Chromium Single Cell 3' Kit. The data was processed and analyzed using the Seurat R package. The study was approved by the local Ethic committee. Results: Patient characteristics: 31 RR DLBCL patients (42% females; median age 72, range 47-81 years), all treated with CAR-T cells therapy as third line of therapy, participated in the study. All patients underwent lymphopheresis, followed by CAR-T production. Disease status at lymphodepletion was complete remission (CR, n=4, 13%), partial remission (n=6, 19%), and progressive disease (n=21, 68%) and patients received either tisagenlecleucel (n=20, 65%) or axicabtagene ciloleucel (n=11, 35%) . PET scan performed on day 30 post treatment revealed that 68% and 32% obtained CR, and PD, respectively. scRNA analysis revealed differences in PBMC composition between healthy donors and LBCL patients (Figure 1A), with lower levels of naïve and memory T cells and elevated levels of activated monocytes in LBCL patients, suggesting a shift towards an inflamed and pre-dysfunctional immune state. Categorization of LBCL patients into responders (PR/CR on day +30 PET-CT) and non-responders (PD on day +30 PET-CT) revealed differences in immune cell subsets between responders and non-responders (Figure 2A), with enriched inflammation-related pathways in non-responders, In contrast, responders exhibited a distinct healthy-like immune profiles, particularly high levels of naive T cells, similar to that obtained in their age matched healthy controls. Of note, non-responders showed alterations in immune cell subsets, including increased levels of T regulatory cells and effector NK cells. Furthermore, non-responders demonstrated a prominent inflammatory immune profile, characterized by gene pathways related to inflammation and activation, such as type I and III IFN-G, and TNFb response (Figure 1B). Notably, an IFN-G score was significantly higher in non-responder's monocytes subpopulations, compared to responders. Conclusions: These findings indicate that the success of CAR-T therapy could be determined by the molecular characteristics and subpopulation distribution of each patient's PBMC. The diverse immune profiles of DLBCL patients might impact the effectiveness of their immune cells in generating successful CAR-T products or providing a conducive environment for CAR T cells to combat cancer. Furthermore, these findings emphasize the potential of scRNA-seq in evaluating PBMC from CAR-T patients prior to treatment, allowing the potential calculation of an “immune potency score” associated with the probability of a positive outcome from CAR-T treatment of DLBCL patients. This score could potentially be used by oncologists in decision-making processes to determine the appropriate candidates for CAR-T therapy.

Functional assessment of DNA extraction methods from frozen human blood samples for Sanger sequencing analysis.

The quality of input DNA is crucial for obtaining significant inferences from molecular techniques like Sanger sequencing and Next Generation Sequencing experiments. Many of the extraction methods are suitable for retrieving quality DNA from fresh blood and tissue samples, regardless of the isolation principle. However, while isolating DNA from frozen blood samples, processed tissue samples or low-quality samples, careful selection of suitable extraction methods is extremely important. Moreover, there is no standard protocol recommended for genomic DNA extraction from stored blood samples, particularly those stored in a Biobank, for applications like Sanger sequencing. Consequently, we have systematically compared different commercial DNA isolation kits with a modified manual extraction method for blood samples frozen for up to three years and assessed their quality, yield and suitability for PCR, Real-Time PCR and Sanger sequencing. The manual DNA extraction method was improved by incorporating a few modifications: a lower NaCl concentration was used for precipitatingDNA and excluded the use of phenol. The modified method provided the maximum DNA yield from stored blood. Although all the methods tested were suitable for recovering DNA from stored blood, the modified method described here may be preferred for large-scale applications as it provides cost-effective ways to obtain large quantities of quality DNA. Most importantly, the DNA isolated by the modified method appears to be more stable in long-term storage at -80°C.

The relationship between unique gut microbiome-derived lipid metabolites and subsequent revascularization in patients who underwent percutaneous coronary intervention

Background and aimsStudies have recently revealed the linoleic acid metabolic pathway of Lactobacillus plantarum, the representative gut bacterium in human gastrointestinal tract, and the anti-inflammatory effects of metabolites in this pathway. However, no clinical trials have evaluated the association between these metabolites and revascularization in patients who underwent percutaneous coronary intervention (PCI). MethodsWe retrospectively reviewed patients who underwent PCI with subsequent revascularization or coronary angiography (CAG) without revascularization. Patients with frozen blood samples at the index PCI and revascularization or follow-up CAG were enrolled. ResultsAmong 701 consecutive patients who underwent PCI, we enrolled 53 patients who underwent subsequent revascularization and 161 patients who underwent follow-up CAG without revascularization. Patients who underwent revascularization showed significantly lower plasma 10-oxo-octadecanoic acid (KetoB) levels (720.5 [551.6–876.5] vs. 818.4 [641.1–1103.6 pg/mL]; p = 0.01) at index PCI. Multivariate logistic regression analysis revealed that decreased plasma KetoB levels at the index PCI were independently associated with subsequent revascularization after PCI (odds ratio; 0.90 per 100 pg/mL increase, 95% confidence interval; 0.82–0.98). Additionally, in vitro experiments showed that the addition of purified KetoB suppressed the mRNA levels of IL-6 and IL-1β in macrophages and IL-1β mRNA in neutrophils. ConclusionsPlasma KetoB level at index PCI was independently associated with subsequent revascularization after PCI, and KetoB could act as an anti-inflammatory lipid mediator in macrophages and neutrophils. The assessment of gut microbiome-derived metabolites may help predict revascularization after PCI.

Abstract 1332: Extracted mRNA from frozen buffy coat samples shows promise for use in downstream sequencing analyses

Abstract Frozen blood samples are routinely collected and stored in medical biobanks across the world, often for periods of several decades, and later used for medical research such as biomarker studies. Some common biomarker targets have included DNA alterations, protein or antibody levels or metabolomics. However, despite the potential importance of gene-expression alterations in circulating immune cells, transcriptional studies on blood samples from biobanks have been limited due to preanalytical technical difficulties of extracting high-quality RNA. In the absence of an RNA preservative, RNA degrades rapidly during the thawing process. Our aim was, therefore, to investigate the possibility of using biobanked blood samples for transcriptomics analyses. First, we used a CryoXtract CXT 350 to establish a protocol capable of isolating high-quality RNA from frozen core aliquots of buffy coat without thawing the entire sample tube. Then, we performed RNA sequencing on RNA extracted from 20 pre-diagnostic buffy coat samples from colorectal cancer patients and matched controls included in the population-based, prospective Northern Sweden Health and Disease Study (NSHDS). For all samples, two different library preparation methods were used (Illumina TruSeq Stranded mRNA poly-A selection and Illumina Stranded Total RNA with Ribo-Zero Globin). We obtained RNA yields of more than 1 µg from a majority of the NSHDS samples (mean=2.57 µg), and more than 92% of samples had RIN values of 6 or higher, indicating suitability for downstream analyses. Preliminary random forest analyses showed expected discriminatory ability between men and women based on data generated from each of the two library preparation methods. In conclusion, our newly established RNA extraction method enables extraction of RNA from frozen, biobanked buffy coat samples, collected in standard tubes, that is of sufficient quantity and quality for use in downstream whole-genome transcriptomic analyses, such as RNA sequencing. Citation Format: Erik Bovinder Ylitalo, Linda Vidman, Sophia Harlid, Bethany Van Guelpen. Extracted mRNA from frozen buffy coat samples shows promise for use in downstream sequencing analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1332.

Investigation of optimal procedures for storage and use of plasma samples suitable for gene doping tests.

Gene doping, which is prohibited in horseracing and equestrian sports, can be performed by introducing exogenous genes, known as transgenes, into the bodies of postnatal animals. To detect exogenous genes, a method utilizing quantitative polymerase chain reaction (qPCR) with a hydrolysis probe was developed to test whole blood and plasma samples, thereby protecting the fairness of competition and the rights of stakeholders in horseracing and equestrian sports. Therefore, we aimed to develop sample storage methods suitable for A and B samples in gene doping tests using blood. For sample A, sufficient qPCR detection was demonstrated after refrigeration for 1 to 2 weeks post collection. For sample B, the following procedures were confirmed to be suitable for storage: 1) centrifugation after sample receipt, 2) frozen storage, 3) natural thawing at room temperature, and 4) centrifugation without mixing blood cell components. Our results indicated that long-term cryopreservation yielded good plasma components from frozen blood samples even though it destroyed blood cells, indicating its applicability to the gene doping test using sample B, which can be stored for later use. Sample storage procedures are as important as detection methods in doping tests. Therefore, the series of procedures that we evaluated in this study will contribute to the efficient performance of gene doping tests through qPCR using blood samples.

PSYCHOSOCIAL AND BEHAVIORAL PREDICTORS OF HEALTH AND AGING

Abstract Crucial to understanding the aging process is to identify modifiable characteristics that can be targeted in hope of optimizing health across adulthood. This symposium brings together a diverse set of talks about the biopsychosocial and behavioral processes that influence aging outcomes. Turiano used a national sample to demonstrate that individuals with higher levels of neuroticism often use food to cope with stressors which ultimately results in a greater central adiposity and elevated glycated hemoglobin. Zavala used an epidemiological sample of long-banked frozen blood samples to successfully extract DNA and methylation biomarkers from 140 adults and found that stress measures are associated with accelerated biological aging. Luo used coordinated analysis to explore how correlations between personality traits and four health outcomes fluctuate over time, and found the associations between personality (level/change) and health increased in strength through middle adulthood and early stage of late adulthood but weakened in very old age. Jackson found that individuals who experienced high and increasing loneliness in older adulthood had worse cognitive function and steeper cognitive decline than expected given their observed post-mortem neuropathology (i.e., worse cognitive resilience). Lastly, Pfund investigated how personality and social support change together and found within and between person associations between personality and social support, and evidence that retirement was associated with an increase in social support. In sum, this symposium presents novel evidence for the associations among personality, stress, social support, and lonliness are associated with physical and cognitive health in old age.

Experimental long-distance haplotyping of OCA2-HERC2 variants

The regulatory HERC2 SNP, rs12913832, is strongly associated with blue and brown eye colour. However, eye colour in heterozygous rs12913832 individuals is observed to vary greatly. Missense mutations in OCA2, such as rs1800407 and rs74653330, are associated with lighter eye colour in some but not all heterozygous rs12913832 individuals. Determining the physical linkage of these variants might help to further explain eye colour variation. So far, experimental haplotyping of these variants has been challenging because the genomic distance between them (∼ 135 kb) exceeds the fragment lengths produced by commonly used DNA isolation kits. The aim for this study was to explore novel methods for long distance haplotyping to assess associations between OCA2-HERC2 haplotypes and eye colour. DNA was isolated from frozen blood samples collected from Norwegians that are known to be heterozygous for both HERC2 rs12913832 and OCA2 SNPs, either rs1800407 (n = 23) or rs74653330 (n = 17), using the newly commercially available Monarch® HMW (heigh molecular weight) DNA Extraction Kit (New England BioLabsinc). We successfully isolated DNA fragments up to 210 kb, which were long enough to haplotype OCA2-HERC2 loci by droplet digital PCR (ddPCR). Three haplotypes were observed in the study population: rs12913832:A-rs1800407:T in 22/23 individuals, rs12913832:A-rs1800407:C in 1/23 individuals and rs12913832:A-rs74653330:T in 16/16 individuals. As expected, all individuals with the rs12913832:A-rs74653330:T haplotype had intermediate to blue eye colour. However, the rs12913832:A-rs1800407:T haplotype was observed in both blue and brown-eyed individuals, suggesting more research is needed.

Prediction of Iron Deficiency Anemia in Third Trimester of Pregnancy Based on Data in the First Trimester: A Prospective Cohort Study in a High-Income Country.

Background: Systematic iron supplementation may be harmful in pregnant women with non-depleted iron. Our objectives were to estimate the prevalence of anemia at the third trimester of pregnancy (T3) and to identify the parameters at the first trimester (T1), which best predict anemia at T3. Methods: This prospective cohort study in France included pregnant women at T1 without non-iron deficiency anemia. Clinical and social characteristics, health-related quality of life, blood count, and a frozen blood sample were collected at T1 and/or T3. Secondly, a matched nested case–control study was built for women with anemia at T3 but not at T1. Multivariate analyses and ROC curves were used to identify the best predictive parameter(s) of anemia at T3. Results: The prevalence of anemia at T3 in the cohort (629 women) was 21.9% (95% CI 18.7–25.2%). In the matched nested case–control study (256 women), hemoglobin (Hb), serum ferritin (SF) and the SF/soluble transferrin receptor ratio at T1 were predictive of anemia at T3 (p < 0.001); however, clinical and social characteristics, as serum hepcidin were not. In multivariate analyses, Hb at T1 was the best predictive biomarker of anemia at T3 with a cut-off value of 120 g/L (specificity 87.5%). Conclusions: The prevalence of anemia at the end of pregnancy remained high in a High-Income Country. Clinical, social, and biochemical parameters did not seem useful to predict anemia at T3 and could not guide iron supplementation. We suggest systematically performing a simple blood count in the first trimester of pregnancy and offering oral iron supplementation for women with Hb < 120 g/L.

Von Willebrand Factor Level and Activity in Correlation with D-dimer Level among COVID-19 Patients in Saudi Arabia

BACKGROUND: Thrombosis and acute respiratory failure are among the leading causes of mortality in COVID-19 patients. The von Willebrand factor (VWF) is one of the main components in the blood coagulation system. It has been hypothesized that the increased VWF level results from vascular damage because VWF is stored in endothelial cells. Infection of endothelial cells with SARS-CoV-2 can stimulate the release of VWF. This study aimed to determine the level of VWF activity among the Saudi population affected by COVID-19 and investigate the correlation between VWF level and D-dimer level. MATERIALS AND METHODS: This is an analytical, retrospective, observational study conducted in a single-center tertiary hospital at King Fahad Military Medical Complex (KFMMC). A study sample of 80 hospitalized patients (aged ≥18 years) diagnosed with COVID-19 confirmed by nasopharyngeal SARS-COV was randomly included in this study. Frozen blood samples were used to measure VWF antigen (Ag) level and activity for all patients. Historical data on hemostasis markers such as platelets (PLTS), fibrinogen, and D-dimer were obtained retrospectively, as well as demographic and clinical data. RESULTS: Data of 80 patients were analyzed to assess VWF Ag level and activity in correlation to D-dimer level. The mean age of the patients was 58.3 (standard deviation 16.18), with nearly two-thirds being females (62.6%). The most common comorbidities were arterial hypertension (67.5%), diabetes (56.3%), and dyslipidemia (22.5%). Only 10% of patients were obese. The mean values of were 3.25 for D-dimer, 3.55 for fibrinogen, 226.4 for PLTS, 9.88 for white blood cell, 237.04 for VWF Ag, 253.21 for VWF activity, and 70.45 for C-reactive protein. Pearson's correlation coefficient revealed that the correlation between D-dimer and VWF Ag and D-dimer and VWF activity was statistically insignificant (P &gt; 0.05). Only six patients had overt thrombosis, but this has no significant correlation with VWF Ag, activity, D-dimer, or platelet count. VWF Ag and activity were higher in intensive care unit (ICU) than non-ICU patients. However, it is not significant statistically. VWF Ag and activity were significantly higher in deceased patients than in survivors. CONCLUSIONS: The results of this study showed that there was no significant correlation between VWF Ag and activity with D-dimer among Saudi patients with confirmed COVID-19.

P-023: 2,3-DIPHOSPHOGLYCERATE DETECTION VIA DIRECT INFUSION HIGH RESOLUTION MASS SPECTROMETRY CORRELATES WITH QUANTITATIVE DETECTION IN BLOOD OF PATIENTS WITH SICKLE CELL DISEASE

Purpose: Sickle cell disease (SCD) is a hereditary and chronic life-threatening disorder, characterized by haemolytic anaemia. Increased 2,3-diphosphoglycerate (2,3-DPG) concentrations, along with decreased oxygen affinity of hemoglobin, may be related to the variability of clinical outcomes in SCD. Furthermore, genomic health data holds promise to improve the prediction of disease severity in SCD. Based on the integration of genomics, metabolomics and clinical data from 1000 SCD patients, to be included in 2022, GenoMED4all aims to develop Artificial Intelligence (AI) based deep learning algorithms to improve the prediction of disease severity and phenotype in SCD. This study aims to correlate non-quantitative metabolomics data obtained from dried blood spots (DBS), one of the inputs for GenoMED4all, to quantitative measurement of 2,3-DPG. Aiming to improve the potential of non-quantitative metabolomics from DBS to asses 2,3-DPG. Materials and methods: In snap frozen blood samples from 37 SCD patients and 29 healthy controls, 2,3-DPG was quantified by liquid chromatography mass spectrometry. 2,3-DPG was also detected in DBS from the same subjects by direct infusion high resolution mass spectrometry (DIHRMS). The oxygen tension at 50% Hb saturation (p50) was determined using a Hemox Analyzer (TCS). Statistical analysis were performed by Spearman’s correlation coefficients (SPSS v26.0.0.1) and Mann Whitney testing (GraphPad Prism v9.3.0). Results: After correcting for Hb, 2,3-DPG concentrations were higher in SCD patients than in controls (p<0.001) and Z-scores for 2,3-DPG, as assessed by DIHRMS, were similar in patients and controls. The Z-scores positively correlated with 2,3-DPG concentrations (Fig1A, 0.353, p=0.004). Because of the anaemia in SCD, red blood cells (RBCs) and plasma make up lower and higher volumes in the blood, respectively, compared to healthy controls. This affects the ratio of RBC and plasma metabolites on the DBS. DIHRMS detects a wide range of RBC and plasma metabolites, whereas the quantitative measurement is restricted to measuring 2,3-DPG of RBCs. To correct for those differences between methods, we applied a correction factor to the DIHRMS data using (1/Ht)*(1-Ht/1), correcting for the RBC volume (1/Ht) and the plasma volume (1-Ht/1). This resulted in a trend towards higher Z-scores in patients than controls (p=0.0597). Moreover, the positive correlation with 2,3-DPG concentrations increased to 0.526 (Fig1B, p<0.001). As 2,3-DPG affects the oxygen affinity of Hb, all measurements were correlated to p50. Expectedly, 2,3-DPG concentrations positively correlated with p50 (0.842, p<0.001). After applying the correction factor to the DIHRMS data, p50 correlations increased from 0.361 (p=0.003) to 0.529 (p<0.001). Conclusion: Strongest correlation between quantitative and non-quantitative methods for 2,3-DPG detection were observed after correcting for both RBC and plasma volumes in non-quantitative metabolomics. After correction, DIHRMS can be used to assess 2,3-DPG concentrations in DBS. This translation of non-quantitative to quantitative metabolomics for 2,3-DPG and potentially other RBC metabolites adds significant value to the use of DIHRMS, as DIHRMS is far more efficient in obtaining extensive information about the total metabolome than quantitative methods. The large population size and the AI based deep learning algorithms of GenoMED4all will enable to evaluate the potential of non-quantitative metabolomics in SCD. Funding: Horizon2020, GenoMED4all(https://genomed4all.eu/), Agios Pharmaceuticals Inc., ERN-EuroBloodNetFig 1. correlations between non-quantitative (Z-score) and quantitative 2,3-DPG detection A) non-quantitative and quantitative 2,3-DPG detection B) non-quantitative detection corrected for RBC and plasma volume and quantitative 2,3-DPG detection The authors do not declare any conflict of interest