EDTA Blood Research Articles

Functional and Multi-Omics Signatures of Mitapivat Efficacy upon Activation of Pyruvate Kinase in Red Blood Cells from Patients with Sickle Cell Disease

2,3-diphosphoglycerate (DPG) in red blood cells (RBCs) stabilizes deoxyhemoglobin to promote hemoglobin (Hb) oxygen off-loading and counteract hypoxia. In sickle cell disease (SCD), mutation of glutamate 6 to valine in the beta chain of Hb results in HbS that polymerizes, causing RBCs to sickle upon deoxygenation in the tissues. Elevated DPG promotes sickling by stabilizing HbS fibers. DPG is an intermediate substrate in the glycolytic pathway in which pyruvate kinase (PK) is a rate-limiting enzyme. In a previous Phase 1 study (NCT04000165, Xu et al, 2022), we established proof-of-concept for activating PK as a viable anti-sickling therapeutic approach; mitapivat, an investigational oral allosteric activator of PK, decreased DPG and increased adenosine triphosphate (ATP) levels, improved hematologic and sickling parameters in patients (pts) with SCD (HbSS). Here, we investigated the changes in the metabolome, lipidome and proteome of RBCs from patients with SCD currently enrolled in an open label, Phase 1/2 study (NCT04610866) that evaluates long-term safety and tolerability of mitapivat in pts with SCD. Fifteen pts with HbSS (aged 25 - 57 years, 10 males) were evaluated. All pts started mitapivat at 50 mg twice daily (BID), escalating after 4 wks to 100 mg BID; dose adjustments were performed for safety and tolerability, per PI discretion. At the time of data cutoff (March 23, 2023), all 15 pts completed the core period of 24 wks, 14 pts completed 48 wks ,10 pts completed 72 wks and 6 pts completed 92 wks. Leuko-depleted RBCs (LDRBCs) obtained from fresh whole blood in EDTA at baseline (V1, prior to drug initiation) and various longitudinal time points over the course of the study were flash-frozen in liquid nitrogen and kept frozen at -80°C until analysis. Clinical measurements of DPG, ATP, red cell PK (PKR) protein and activity levels, oxygen affinity (p50) and sickling kinetics (t50) were accompanied by multi-omics (metabolomics, lipidomics, proteomics) characterization of the LDRBCs. In total, 150 (6x12, 4x10, 4x8 and 1x6) timepoint samples were analyzed (Fig 1A). Multi-omics analyses were performed through a stepwise extraction of metabolites, lipids and proteins on 50 µl of packed LDRBC pellets. Extracts were separated via ultra-high-pressure liquid chromatography coupled to mass spectrometry, prior to peak picking and identification of ddMS2 data as previously described (Thomas et al 2020). Statistical analyses were conducted using MetaboAnalyst v 5.0 and Rstudio upon normalization of relative quantitative data to visit 0 levels for each variable. Network analysis and pathway analysis were performed in OmicsNet v 2.0. Mitapivat therapy had significant effects on the metabolome, lipidome and proteome of SCD RBCs. Overall, the temporal trends of omics responses to mitapivat treatment across visits was evident, especially when focusing on the top 50 metabolites/lipids or proteins by time series ANOVA. Specifically, results suggested a positive correlation between mass spectrometry-based quantitation of RBC mitapivat levels and DPG consumption, ATP accumulation, PKR activity (measured via enzymatic assays), and PKR protein levels, with internal consistency between omics data and antibody-based or enzymatic assays (Fig. 1A). The treatment also promoted activation of the Lands cycle, transient elevation of lysophosphatidylcholines and oxylipins, and depletion in free fatty acids within the first 6 months, suggestive of an effect on membrane lipid remodeling. SCD RBCs abnormally retain mitochondria; here, we showed a consistent decrease in the levels of residual mitochondrial proteins in RBCs within 4 wks of mitapivat treatment (Fig. 1B). In keeping with the predicted activation of PKR upon mitapivat treatment, we observed decreases in DPG and increases in ATP, with elevation in glycolytic fluxes, as inferred from longitudinal changes in glycolytic intermediates at the hexose and triose phosphate steps, as well as lactate. Unsupervised analyses of omics data confirmed the specificity of mitapivat on targeting late glycolysis, with glycolytic metabolites ranking as the top correlates to parameters of oxygen affinity (p50) and sickling kinetics (t50) during mitapivat treatment. Multi-omics analysis of RBCs from HbSS patients treated with mitapivat identified benefits for glycolysis, DPG consumption and ATP generation, as well as activation of the Lands cycle.

Evaluation of Red Cell Deformability in Sickle Cell Disease Patients, Sickle Cell Carriers, and Healthy Controls Using Lorrca-Derived Parameters

BACKGROUND Red blood cell (RBC) deformability plays a critical role in RBC elongation and viscosity, and when reduced, perfusion of peripheral tissues and oxygen delivery are impaired. Intracellular ATP maintains water and ion homeostasis in RBCs; reduced ATP leads to water and ion loss, dehydration and loss of RBC elasticity. RBC dehydration increases mean corpuscular hemoglobin concentration (MCHC) and also affects distribution of RBC width (RDW). Patients with sickle cell disease (SCD) have reduced RBC ATP, and markedly reduced RBC deformability when compared to healthy individuals. Here, we employed Laser-Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics) to evaluate deformability in RBCs from patients with SCD (HbSS) compared to RBCs from sickle cell carriers (HbAS) and ethnic-matched healthy controls (HbAA). We further analyzed how these LORRCA-derived parameters correlate with ATP, MCHC, and RDW. METHODS We enrolled adult subjects (age ≥ 18 years) of African-descent, and not recently transfused (within 8 weeks) under protocol NCT03685721 approved by the NHLBI Institutional Review Board. Fresh whole blood in EDTA was processed on the same day of collection. Cells were counted and diluted in polyvinylpyrrolidone (PVP) solution before undergoing LORRCA assays including deformability, osmoscan, and oxygenscan (HbSS subjects). The deformability assay uses the elongation Index (EI) to measure the cells' ability to undergo deformation with increasing shear stress at 0.95, 3, and 30 Pascals (Pa). The osmoscan assay measures the deformability under continuous osmotic changes (0-600mOsm/kg) and has several parameters: O min (RBC fragility); El max (maximum deformability and membrane flexibility); O max (osmolality where EI max is achieved) and O hyper (intracellular viscosity and maximum deformability in the hypertonic region). The oxygenscan assay measures the oxygen pressure at the time when RBCs start to become rigid (point of sickling). Whole blood levels of ATP were measured using LC-MS/MS with LLOQ at 50.0 μg/mL and converted to intracellular concentrations by dividing by the hematocrit (as a fraction). Descriptive statistics and correlation (Spearman) of ATP, RDW, and MCHC with each LORRCA parameter were performed using R (v4.2.3) and Prism(v9). RESULTS We studied 156 subjects which comprised of 63 HbSS (33 males), 61 HbAS (23 males) and 32 HbAA (8 males) genotypes. The average age in years (range) in HbSS was 32 (18-58); in HbAS, 43 (18-70), and in HbAA, 38 (19-72). As shear stress and osmotic pressure increased, the mean EI and osmolality were significantly (p<0.001, unpaired two-sample t-test) lower in the HbSS cohort compared to HbAS and HbAA (Figure 1). There was no significant difference in the mean EI and osmolality between HbAA and HbAS with increasing shear stresses and osmotic pressures. As shear stress increased, the correlation of deformability with reduced ATP got significantly stronger in the HbSS cohort (r= -0.351 at 0.95 Pa to r=-0.401 at 30 Pa) but the trend was reversed for the HbAS and HbAA cohort (Table 1). Under increasing osmotic pressure, a significant correlation was found between reduced ATP and maximal deformability (EI Max) in HbSS (p=0.008) compared to HbAS (p=0.896) and HbAA (p=0.114). MCHC was significantly correlated with RBC hydration state (O Hyper) in all 3 genotypic groups (HbSS, HbAS and HbAA). Similarly, RDW correlated with maximal deformability (EI Max) under increasing osmotic pressure in all 3 cohorts. In the HbSS subjects, there was a significant positive correlation of ATP and RDW with point of sickling (oxygenscan); MCHC was negatively correlated but was not significant. CONCLUSION RBC deformability was reduced in HbSS patients compared to HbAS and HbAA under increasing shear stress and osmotic pressure. ATP levels showed significant correlation with RBC deformability as measured by LORRCA, particularly in patients with SCD, suggesting increasing ATP levels as an important therapeutic strategy in patients with SCD. In keeping with published data, our analyses showed that RBC volume and size as well as intracellular hemoglobin concentration can affect deformability.

Five Novel Hemizygous Variants in the PGK1 Gene Associated with Neurological Dysfunction and Mild Hemolytic Anemia in Five Indian Patients

Introduction Phosphoglycerate kinase (ATP: 3-phosphoglycerate 1-phosphotransferase; EC 2.7.2.3; PGK) is the crucial enzyme of the glycolytic pathway, catalyzing1, 3 bis-phosphoglycerate to 3-Phosphoglycerate, generating one molecule of ATP [1]. PGK enzyme is encoded by the PGK1 gene located at Xq13.3, measures 23kb in size, and spans into 11 exons. PGK deficiency is an X-linked disorder associated with a very rare cause of hereditary non-spherocytic hemolytic anemia (HNSHA). PGK1 gene is ubiquitously expressed but the patients exhibit three major symptoms -1) haemolytic anemia 2) myopathy (rhabdomyolysis), 3) mental retardation and various neurological manifestations[1]. The severity of symptoms varies among patients with PGK deficiency. PGK deficiency is reported worldwide, although to our knowledge no case with Indian ancestry has been reported. Method Genomic DNA was extracted from the EDTA blood sample and processed for targeted Next Generation Sequencing (t-NGS) as per the manufacturer's protocol. The t-NGS panel broadly consists of 80 genes of the spectrum of the disorder including congenital hemolytic anemia, bone marrow failure disorders, and iron deficiency anemia. The libraries were sequenced to >80-100X mean coverage on the Illumina sequencing platform. The variants were classified based on ACMG guidelines. The impact of non-synonymous variations was estimated using PolyPhen-2, SIFT, MutationTaster2, CADD Phred, and Mutpred2. In silico effect of the variant was studied by PyMol. Latter the enzyme deficiency was confirmed by standard protocol and parental segregation analysis was performed. Relative quantification (qPCR) was evaluated based on the comparative CT method ( 2 − ΔΔCT). Result We report five male children of <4 years of age with PGK deficiency from Indian origin. Clinically, all of them manifest global development delay and no speech. Autism spectrum disorder is observed in two of the cases, incidence of seizure and convulsion are present in two cases. None except one, presented with haemolytic anaemia and was given one transfusion post an episode of seizure at the age of 2 years. Additionally, a family history of haemolytic anemia with developmental delay and seizure was present in only one case although the patient himself required no transfusion requirement to date. All the patients are routinely followed up for any evidence of a haemolytic crisis, transfusion requirement, or any other clinical symptoms. Detailed phenotypes of all the patients are listed in Table 1. t-NGS revealed five novel hemizygous variants in the PGK1 gene - p. Cys50Trp, p. Gly137Trp, p. Val140Ile, p. Asp159Asn, and p. Arg330Trp. These variants have not been reported in the literature or available databases. DNA Sanger sequencing confirmed the heterozygous state in the mothers. In silico tools confirmed the pathogenicity of the variants. The PGK enzymatic activity was markedly reduced. The PGK1 transcript levels were reduced to 60% in patients as compared to the healthy controls. Discussion To date, only 34 mutations are known to be associated with PGK enzyme deficiency. Amongst them, 50% of variants exhibit symptoms of anaemia whereas the remaining show neurological and /or myopathy. Figure 1 illustrates all 39 mutations reported with PGK deficiency. Only four mutations p.E120K, p.G158R, p.A354P, and p.I371K are known to exhibit all three categories of symptom (PGK-anaemia-neurological-myopathy)[2]. Conclusion NGS allows for the identification of novel genetic variants that may contribute to the development of rare disorders, expanding our knowledge of their underlying mechanisms. This advanced technology also enables researchers to analyze multiple genes simultaneously, increasing the efficiency and speed of diagnosis in unexplained cases of HNSHA.

Study prognostic significance of low platelet count in newborn admitted in neonatal intensive care unit (NICU) at tertiary care center

One of the most frequent haematological abnormalities found in new-borns is Thrombocytopenia. In neonates admitted to ICUs, thrombocytopenia develops in 18–35 % of all patients. With decreasing gestational age and birth weight, the incidence are increasing. Platelets are formed by megakaryocytes and are present in the bloodstream for 5–7 days. Platelets are known as regulators of haemostasis and thrombosis. Platelets become active in the blood resulting vascular injury. Thrombopoiesis is the formation of platelets in the Bone marrow. Thrombopoietin is the main regulator of thrombopoiesis. Thrombopoietin affects most aspects of the production of platelets. The process of Thrombopoiesis is caused by the breakdown of proplatelets (mature megakaryocyte membrane pseudopodia projections). Aim is to assess the prevalence, causes, treatment modalities, and prognostic outcomes of thrombocytopenia in neonates. Research design was carried out in the Department of paediatrics and neonatology, Government Medical College, Surat from October 2020 to April 2022. Data were collected from MRD section. Platelet numbers were estimated from whole blood EDTA sample taken from neonates for routine medical management. Data were analysed for Age, Sex, Intraventricular haemorrhage, other bleeding manifestation, Necrotising enterocolitis, Sepsis, Fungal infection & final outcome. Out of 1265 neonates admitted to the NICU at Department of paediatrics and neonatology, GMC, Surat 450 neonates were found to have thrombocytopenia. Male neonates are more significantly affected than female neonates. The early platelets drop even without the later development of thrombocytopenia is an early indicator of poor outcome and major morbidities, mainly infection.

Assay of macrotroponin I not complexed with troponin T: effect on the performance of troponin T to predict myocardial infarction

Abstract Background Cardiac troponin-immunoglobulin (Ig) complexes (macrotroponin) can interfere with cardiac troponin (cTn) test performance. However, the role of this interference has not been fully explored. We developed a specific, novel, 2-hour prototype immunoassay of monomer IgG-bound TnI (bTnI-IgG). Using this assay, our purpose was to examine the potential contribution of cTnT binding to macro-TnI, and report its effects on the clinical diagnostic and predictive performance of highly sensitive TnT (hs-TnT) and TnI (hs-TnI) assays. Methods Using the bTnI-IgG assay, we evaluated EDTA blood samples taken at presentation (t=0) from two ED acute chest pain studies; SPACE (n=1066) and FAST-TRAC (n=1440), with statistical analysis (logistic regression, ROC curves), to AMI and 1-year outcomes, alone and in combination, with hsTnT (Roche) and hsTnI (Abbott/Beckmann) tests. Pathway analysis was used to identify if single bTnI-IgG measurements improve risk stratification in low and high AMI risk. Results In individual and combined study datasets, bTnI-IgG concentrations in patients with adjudicated acute MI (n=336) were lower than all other diagnoses (p<0.001). Singularly, and in multivariable models containing hs-TnT or hs-TnI concentrations, bTnI-IgG was an independent predictor of MI. Amongst all patients with MI not ruled-out on presentation (hs-TnT >5ng/L) and not high-risk (hsTnT<14ng/L), the addition of bTnI-IgG to a model containing hs-TnT, HxMI and sex improved discrimination (AUC increase of 0.014 (95%CI: 0.004 to 0.025) from 0.788 to 0.802, p=0.009). Within a diagnostic pathway analyses of n=2126 matched patient data points, adding presentation bTnI-IgG measurement alone to an MI risk stratification model consisting of hs-TnT ≤URL, HxMI and sex yielded 14% more low risk patients identified (n=586 v 667 patients) through improved specificity (77% to 88%) whilst maintaining an overall sensitivity of >99%. bTnI-IgG also predicted 1-year mortality and new stroke in all patients (Odds Ratios 0.292 [95%CI 0.130 to 0.654] p=0.009) and 0.255 ([95%CI 0.078 to 0.834] p=0.024), respectively. Conclusions Measurement of bTnI-IgG that is free from cTnT could potentially improve ED presentation diagnostic test performance and risk stratification of MI patients using criteria that includes hs-TnT. No effects upon hs-TnI assays were observed. This data suggests current hs-TnT test design and measurements may be impacted by binding interactions between TnT and TnI. Finally, bTnI-IgG can independently predict mortality and stroke outcomes within 1 year of index presentation.

Streamlining the Detection of FLT3 Internal Tandem Duplication (FLT3-ITD) Using the Cepheid Genexpert® Automated System

Objectives: The development of targeted therapies for Acute Myeloid leukemia (AML) demands fast and reliable diagnostic tests. Specifically, FMS-like tyrosine kinase 3 (FLT3-ITD), comprising nearly 25% of all AML cases, is the most common targetable mutation in AML. Current diagnosis of FLT3-ITD by PCR involves numerous laborious steps, with bench-top steps involving time-consuming whole blood processing, DNA isolation, plate-based PCR and subsequent fragment analysis by capillary electrophoresis. Consequently this adds significant overhead, time-consuming personnel training, and numerous steps which could lead to errors, all of which increase the resource burden and time to result. The objective of this study was to create an automated FLT3-ITD diagnostic prototype that is faster, requires less hands-on time that can detect all clinically relevant ITD lengths. Methods: We have developed a simplified FLT3-ITD prototype assay using the Cepheid GeneXpert® automated platform for FLT3-ITD detection. The prototype substantially streamlines the process, using a less than 30 minutes off-board time to prepare a universal blood lysate that is compatible with all Cepheid Hematology Oncology products. Once the sample is loaded into the cartridge and inserted into the instrument, the cartridge performs automated DNA isolation, purification, and qPCR. A Taqman-based probe for ABL is utilized in the qPCR step as a PCR control to ensure that the blood processing and qPCR was successful and avoid wasted downstream steps. Quantitative fragment analysis can easily be performed on the PCR product using capillary electrophoresis (CE) for precise allelic ratio (AR) callout. Additionally, the PCR product is compatible be run on a bioanalyzer or standard Agarose Gel-Electrophoresis if available. Results: The developed automated FLT3-ITD diagnostic prototype demonstrated significant improvements over the existing methodologies with substantially reduced time to result and simplicity in use. To assess the sensitivity of this prototype, we used MV4-11 cancer cells harboring a 30 bp ITD and CRISPR-CAS engineered human cell lines with 3 bp, 21 bp, and 75 bp FLT3 ITD insertions, comprising the most commonly detected ITD lengths spiked into EDTA blood as a test material. For these ITD's, we observed sensitivity and linearity down to 0.01-0.02 by CE for the ITDs tested. Importantly, the prototype detected all lengths of ITD mutations with inserts from 3bp to 300 bp using plasmid test materials. Conclusion: We have successfully developed a novel FLT3-ITD diagnostic prototype that has several major advantages over existing methodologies. The prototype significantly reduces the time and hands-on effort required for analysis, streamlining the diagnostic process for same day results. The larger sample volume used allows improved sensitivity to ensure accurate detection, even at low allelic frequencies. Additionally, the prototype requires fewer reagents in that sample prep and qPCR are performed in a single cartridge and run hands-free. Downstream analysis of amplified products can be easily performed and validated using capillary electrophoresis, bioanalyzer, or gel electrophoresis. Finally, because this prototype uses the same universal blood lysate as other AML tests, it has the potential to improve future diagnostic therapeutic interventions in the clinic.

Inclacumab Reduce Preexisting Red Blood Cell Adhesion to Activated Endothelial Cells: In-Vitro Assessment of the Microfluidic Platform Endothelium-on-a-Chip

Background: In 2019 FDA approved the use of an antibody anti P-selectin (Crizanlizumab) for the treatment of sickle cell disease (SCD) to reduce the vaso occlusive crisis (VOCs). New anti-adhesive treatments are being developed recently. Global Blood Therapeutics, Inc., acquired by Pfizer Inc. in October 2022, acquired Inclacumab from Roche and started two clinical trials now in phase 3 to evaluate the capacity of this humanized IgG4 monoclonal antibody anti P-selectin to reduce the VOCs in patients with SCD. In our recent work presented at EHA 23 in Frankfurt Germany, we showed that Inclacumab prevents the adhesion of RBCs to chronically and acutely heme activated human umbilical vein endothelial cells (HUVECs) with a statistically significant higher effect in comparison to Crizanlizumab. In this work we used a similar approach to test the capacity of Inclacumab in the reduction of preexisting adhesion events on acutely heme activated HUVECs. Methods:Whole blood EDTA samples from six SCD subjects all with HbSS genotypes were collected at University Hospital Cleveland Medical Center, Cleveland, OH, USA. To standardize the samples, we isolated RBCs by centrifugation and subsequential washes in Phospahte buffer s of the whole blood and resuspended in basal cell culture medium (EBM; Lonza, Morristown, NJ, USA) at a hematocrit of 20% with 10 mM of HEPES. HUVECs (Lonza, Morristown, NJ, USA) were introduced and maintained within the endothelialized microfluidic channels at physiological flow for at least 48-72 hours prior to experiments. We performed an acute short-term activation, where the blood samples were supplemented with 40 µM heme +/- 100 µg/ml injected through the microfluidic channels for 15 minutes, after the adhesion took place we washed the non-adherent cells with Inclacumab and Crizanlizumab and we compared with untreated washing buffer (both compounds were provided by Global Blood Therapeutics, Inc., a wholly owned subsidiary of Pfizer Inc. South San Francisco, CA, USA) A phase-contrast images of the remaining RBCs were acquired with an inverted microscope (DMi8 Leica Microsystems Inc. Deerfield, IL, USA) and quantified based on previous published methods (Figure 1A). Paired t-test was used to calculate statistical significance. Results: Acute short-term heme activation induces high level of RBCs adhesion. Both compounds Inclacumab and Crizanlizumab showed a reduction of the number of adherent RBCs to the activated heme, but only Inclacumab results statistically a significant higher reduction compared to untreated RBCs (Figure 1B). Conclusions: Inclacumab is able not only to prevent the adhesion of RBCs to acutely and chronically activated HUVECs but is able to remove the preexisting adhesion and results in significant higher reduction compared to untreated. RBCs adhesion assay on Endothelium-on-a-chip is a novel and efficient way to study the efficacy of the new antiadhesive drug and could be used to monitor patient response to new therapies in SCD. Figure1 : Effect of Inclacumab vs Crizanlizumab on removal of SCD RBC adhesion to heme activated HUVECs. A) Schematic of adhesion experiment and inverted microscope contrast of phase images 10X of adherent RBCs at acutely heme activated HUVECs. B) Inclacumab has statistically significant effect on remove adherent RBCs to acutely heme activated HUVECs (p=0.009, N=7) For all experiments, the heme concentrations were 40 µM and Inclacumab and Crizanlizumab were 100 µg/ml. Scale bare 20 µm. P-values were based on paired t-test. Error bars represent the standard error of the mean (SEM)

Assessing the diagnostic potential of 16SrRNA gene for neonatal sepsis: A tertiary care hospital study in South India

BackgroundThe need of the hour is to incorporate a rapid assay that could efficiently detect neonatal sepsis. We evaluated the diagnostic utility of 16SrRNA broad-range polymerase chain reaction (PCR) in neonatal sepsis. MethodsThe demographic and clinical details of 100 neonates clinically suspected to have sepsis were collected adopting pretested clinical proforma, followed by baseline laboratory investigations, including blood culture, complete blood counts, and C-reactive protein (CRP). Around, 0.2–0.3 ml of the EDTA blood was subjected to enrichment followed by DNA isolation using the modified spin column method. Based on the blood culture report, neonates were further divided into the suspected sepsis group (n = 50) and the confirmed sepsis group (n = 50). We performed 16SrRNA broad-range PCR to identify the presence of bacteria, and the results were analyzed statistically using SPSS software. ResultsNeonates in both groups were found to have clinical parameters comparable to each other except for birth weight, length, and head circumference, which was found to be lower in the culture-positive group than in culture-negative group (p < 0.05). The diagnosis of neonatal sepsis by 16SrRNA broad-range PCR compared to blood culture revealed 100% sensitivity, 64% specificity, and 73.5% positive and 100% negative predictive value. The 18 cases detected positive by PCR had clinical and other diagnostic findings consistent with sepsis. ConclusionThe 16SrRNA broad-range PCR effectively ruled out sepsis in 32 neonates within 8 h of sample collection compared to blood culture, which took 24 h. The method may not replace blood culture but can be used to complement it.

Analysis of genome-wide 5-hydroxymethylation of blood samples stored in different anticoagulants: opportunities for the expansion of clinical resources for epigenetic research

ABSTRACT Background: Elucidating epigenetic mechanisms could provide new biomarkers for disease diagnosis and prognosis. Technological advances allow genome-wide profiling of 5-hydroxymethylcytosines (5hmC) in liquid biopsies. 5hmC-Seal followed by NGS is a highly sensitive technique for 5hmC biomarker discovery in cfDNA. Currently, 5hmC Seal is optimized for EDTA blood collection. We asked whether heparin was compatible with 5hmC Seal as many clinical and biobanked samples are stored in heparin. Methods: We obtained 60 samples in EDTA matched to 60 samples in heparin from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. Samples were comprised of 30 controls and 30 individuals who were later diagnosed with colon cancer. We profiled genome-wide 5hmC in cfDNA using 5hmC-Seal assay followed by NGS. The 5hmC profiling data from samples collected in EDTA were systematically compared to those in heparin across various genomic features. Results: cfDNA isolation and library construction appeared comparable in heparin vs. EDTA. Typical genomic distribution patterns of 5hmC, including gene bodies and enhancer markers, were comparable in heparin vs. EDTA. 5hmC analysis of cases and controls yielded highly correlated differential features suggesting that both anticoagulants were compatible with 5hmC Seal assay. Conclusions: While not currently recommended for the 5hmC-Seal protocol, blood samples stored in heparin were successfully used to generate analysable and biologically relevant genome-wide 5hmC profiling. Our findings are the first to support opportunities to expand the biospecimen resource to heparin samples for 5hmC Seal and perhaps other PCR-based technologies in epigenetic research.

Haemato-biochemical characterization of equine piroplasmosis asymptomatic carriers and seropositive, real-time PCR negative horses

Equine piroplasmosis (EP) is caused by Theileria equi and Babesia caballi, transmitted by tick vectors. Horses can suffer an acute, subacute, and chronic forms of the disease, with clinical signs such as poor performance, fever, pale mucosal membranes, and jaundice. The diagnosis of EP subclinical cases is complex due to the sensitivity of real-time PCR and the limited parasite load in some carriers, making it challenging to differentiate them from seropositive, PCR negative (S+PCR-) individuals. This study aimed to describe haematological and biochemical changes in asymptomatic EP carriers, EP S+PCR- horses and control horses (EP seronegative and PCR negative). It also investigated potential haemato-biochemical markers to aid in distinguishing true EP carriers alongside molecular and serological tests. A comprehensive haematology and biochemistry profile was conducted on 410 sera and EDTA blood samples, comprising 130 EP positives by real-time PCR and competitive ELISA (cELISA) (carriers), 130 EP negatives by real-time PCR but positive to cELISA (S+PCR-) and 150 EP negative horses to real-time PCR and c-ELISA (controls). Our study confirmed that a haematological and biochemistry profile could help to differentiate between EP carriers/S+PCR- from healthy horses. Carriers and S+PCR- horses showed significant increases in the white blood cell count (WBC), high total proteins (TP) and total globulins (GLOB) concentration, and liver function markers compared to controls. Additionally, the evaluation of uric acid (UA) suggested oxidative stress in carrier horses. However, no useful haemato-biochemical diagnostic markers were identified to aid the challenging differentiation of EP carriers and S+PCR- horses, highlighting the need for improvement in molecular/serological diagnosis for these horses.

B-265 Prevalence of 844ins68 Polymorphism in the Cystathionine β-Synthase Gene in Brazilian Individuals

Abstract Background Deficiency of Cystathionine β-Synthase (CBS) is the most common cause of the homocysteine increase in blood and urine. This is an important autosomal recessive disorder with pleiotropic clinical manifestations in the vascular system, the skeletal structure, the ocular lens, and the central nervous system. Several polymorphisms in CBS gene have been associated with homocystinuria, with consequent premature arterial and/or venous occlusive diseases. The thymine-to-cytosine substitution at nucleotide 833 of the CBS gene (T833C) is the majority predominant mutation in individuals with homocystinuria. Several studies of T833C have been performed on patients where venous or arterial occlusive diseases are an important risk factor. The T833C causes a substitution of hydrophobic isoleucine by a more hydrophilic threonine at codon 278 (I278T) and affects the CBS conformation or the interaction of the CBS subunits. Most occurrences of T833C are known to segregate in cis with 844ins68 insertion polymorphism in the exon 8, duplicating the intron 7 splice acceptor. At the mRNA level, polymorphisms T833C and 844ins68 are skipped next to the use of this alternative splicing site. Therefore, previous studies about 844ins68 produced conflicting results that show a neutral or even effect protective of the insertion against occlusive venous and arterial occlusive disease. Thus, this study aims to describe the genotypic profile of the 844ins68 mutation in CBS gene in Brazilian individuals affect with vascular diseases or patients at risk for genetic variants segregating in the family. Methods The study group consisted of 1301 individuals submitted to the identification of the CBS 844ins68 in Grupo Pardini Laboratory. Genomic DNA was isolated from EDTA blood using QIAcard FTA Classic (Qiagen). To detect the insertion, the genomic fragments comprising the intron 7 and exon 8 in the coding region and splicing junctions of the CBS gene were amplified by Polymerase Chain Reaction (PCR). Results Our sampling included 214 men’s blood samples (16.4%) and 1087 women’s blood samples (83.5%). We found a prevalence of 21.6% individuals with the insertion. Males without the 844ins68 mutation (wild type) predominated 161/214, (75.2%), carriers of 844ins68 heterozygotes were 45/214 (21%), and homozygotes were 8/214 (3.7%). In female individuals, was found 859/1087 (79%) allele without 844ins68 (wild type), 214/1087 (19.7%) carriers of 844ins68 heterozygotes and 14/1087 (1.3%) homozygotes. The number of solicitations for the exam in women is greater compared to men. Conclusions Our findings show that the prevalence of 844ins68 in Brazil is compatible with others studies involving Brazilian women with cardiovascular defects. According to the literature, the insertion is most frequent in females than in males. Considering the possible restoration of non-mutated RNA through preferential splicing of the 844ins68, our study suggest that mutated individuals have the insertion as a protective factor for the development of venous or arterial occlusive diseases. The search of 844ins68 should be evaluated in association with the clinical aspects.

IGF1 Genetic Polymorphism and the Association between Vitamin D Status and BMI Percentiles in Children.

Both the IGF1 axis and hypovitaminosis D play a role in childhood obesity, either as a cause or a causality. While some studies suggest an interrelation between vitamin D status, IGF1, and obesity, this mechanism remains obscure. The aim of this study, therefore, was to explore associations between four genetic polymorphisms in the IGF1 axis in hypovitaminosis D-related obesity. The study included 116 pre-pubertal Israeli Arab children (52 girls), mean age 9.4 ± 2.6. Serum 25(OH)D was measured and anthropometric measures were obtained. Genomic DNA was extracted from peripheral EDTA-treated anti-coagulated blood using a standard protocol. Genotypes were determined using the Taqman allelic discrimination assay. The IGF genetic score was computed according to the additive genetic score model. A moderate-to-high negative correlation (r = 0.580, p < 0.05) was seen between the vitamin D status and body mass index (BMI) percentile of participants with high GS. Yet, no correlations were seen between vitamin D status and BMI percentile for participants with a low-to-moderate genetic score (GS) (GS ≤ 2). These results suggest that IGF1 genetic scores associated with elevated circulating IGF1 may indicate a tendency toward developing hypovitaminosis D-associated obesity.

Evaluating transport conditions of conventional, widely used EDTA blood tubes for gene expression analysis in comparison to expensive, specialized PAXgene tubes in preparedness for radiological and nuclear events

Purpose Gene expression (GE) analysis of a radio-sensitive gene set (FDXR, DDB2, WNT3, POU2AF1) has been introduced in the last decade as an early and high-throughput prediction tool of later developing acute hematologic radiation syndrome (H-ARS) severity. The use of special tubes for RNA extraction from peripheral whole blood (PAXgene) represent an established standard in GE studies, although uncommonly used in clinics and not immediately available in the quantities needed in radiological/nuclear (R/N) incidents. On the other hand, EDTA blood tubes are widely utilized in clinical practice. Material and Methods Using blood samples from eleven healthy donors, we investigated GE changes associated with delayed processing of EDTA tubes up to 4 h at room temperature (RT) after venipuncture (simulating delays caused by daily clinical routine), followed by a subsequent transport time of 24 h at RT, 4 °C, and −20 °C. Differential gene expression (DGE) of the target genes was further examined after X-irradiation with 0 Gy and 4 Gy under optimal transport conditions. Results No significant changes in DGE were observed when storing EDTA whole blood samples up to 4 h at RT and subsequently kept at 4 °C for 24 h which is in line with expected DGE. However, other storage conditions, such as −20 °C or RT, decreased RNA quality and/or (significantly) caused changes in DGE exceeding the known methodological variance of the qRT-PCR. Conclusion Our data indicate that the use of EDTA whole blood tubes for GE-based H-ARS severity prediction is comparable to the quality of PAXgene tubes, when processed ≤ 4 h after venipuncture and the sample is transported within 24 hours at 4 °C.

A-375 Evaluation of the CLIA-Waived Afinion HbA1c Point-of-Care Test

Abstract Background Point-of-care (POC) HbA1c tests can be essential in evaluation/monitoring of diabetes provided that the test has acceptable analytical performance. The Abbott Afinion HbA1c is an in vitro diagnostic test for quantification of HbA1c in human capillary and venous whole blood. This study aim was to evaluate the performance of Afinion HbA1c in whole blood EDTA patient samples. Methods The Afinion utilizes the boronate affinity principle for measurement of HbA1c. Hemoglobin A1c was tested on the Abbott AfinionTM 2 analyzer. Two levels of quality control (QC) and two levels of whole blood EDTA patient samples (one low and on high) were used to assess imprecision. Within-day precision was determined by assaying the 20 times in one day and between-day precision was determined by assaying the samples in duplicates over a period of 10 days. The analytical measured range (AMR) and method comparison was performed by using whole blood EDTA patient samples. Percent bias plot and Deming regression analysis was performed by EP Evaluator. Results Within-day and between-day precision and accuracy data are summarized in table 1 below. The assay displayed good linearity over the measured AMR. Lastly, the method comparison between Afinion vs Cobas pro, had a correlation coefficient R2 of 0.994, slope (m) of 1.14 and % bias was 2.5%. Afinion vs Trinity Biotech HPLC had R2 = 0.996, m = 1.04 and % bias was −2.3%. Afinion vs DCA Vantage had R2 = 0.991, m = 1.03 and % bias 1.5. Conclusion We conclude that the Abbott Afinion POC device evaluated here is precise and yields results consistent with other Clinical Chemistry analyzers such as Cobas Pro and Trinity Biotech.

THE ROLE OF A GENOMICS CLINICAL NURSE SPECIALIST IN HELPING TO BUILD A PATHWAY FOR WHOLE GENOME SEQUENCING TO BE OFFERED TO NEURO-ONCOLOGY PATIENTS

Abstract AIMS Whole genome sequencing is currently being offered to paediatric, teenage and young adult neuro-oncology patients and this was being managed by a genomics practitioner who works cross-site. We found that some patients are being discharged from the hospital without 1 component of the test or the consent. Our aim is to create a solid pathway in which fresh tissue, blood sample and record of discussion are taken at a certain stage in their hospital admission. METHOD A poster was created to remind the theatre team that a fresh tumour tissue and EDTA blood sample are the required samples to proceed with whole genome sequencing. Consent training is being provided to all clinical nurse specialists who look after CNS tumours in order to secure the record of discussion at any point in their hospital appointment. After several shadowing and doing the consenting process with supervision, they are expected to be able to be independent in doing this. RESULTS The clinical nurse specialist team have consented 24 out of 37 paediatric, teenage and young adult neuro- oncology patients that were operated since July 2022. Almost all of them have had their bloods taken intra- operatively with some taken whilst still an in-patient. CONCLUSIONS Whole genome sequencing will soon be offered to all neuro-oncology patients and the entire team will need to be equipped in securing the record of discussion in order for this test to proceed and this will be led by the clinical nurse specialist team.

Utility of a microRNA panel in diagnosis and prognosis of hepatitis C-associated hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the leading cause of cancer mortality. Various studies have linked dysregulated microRNA expression to liver cancers, but those related to viral hepatitis-related HCC are limited. We investigated the diagnostic and prognostic roles of circulating miR-331-3p, miR-23b-3p, and miR-3194-5p in EDTA-treated blood samples of 50 hepatitis C virus (HCV) HCC patients, 50 HCV cirrhotic patients, and 50 healthy controls using quantitative real-time polymerase chain reaction. We found that miR-23b-3p and miR-3194-5p were significantly downregulated, whereas miR-331-3p was upregulated in HCC patients compared with controls. Also, these miRNAs were significantly dysregulated in HCC compared with cirrhotic patients. For the diagnosis of HCC, miR-331-3p and the combined miRNAs panel had the highest area under the curve (AUC), followed by miR-3194-5p. The highest AUC for differentiating metastatic from nonmetastatic patients was shown by miR-331-3p and the combined miRNAs panel, followed by miR-23b-3p. Dysregulation of miRNAs was associated with poor clinicopathological manifestations. Finally, miR-331-3P was found to be an independent risk factor for metastatic lesions in HCC. Overall, the assessed miR-331-3p, miR-23b-3p, and miR-3194-5p were significantly associated with poor clinicopathological features of HCC and could be used to discriminate HCV-related HCC patients from cirrhosis and differentiating metastatic from nonmetastatic patients, primarily miR-331-3p along with combined miRNAs. Moreover, miR-331-3p was found to be an independent factor for metastatic lesions.

Analyzing Flow Cytometry or Targeted Gene Expression Data Influences Clinical Discoveries-Profiling Blood Samples of Pancreatic Ductal Adenocarcinoma Patients.

Monitoring the therapeutic response of pancreatic ductal adenocarcinoma (PDAC) patients is crucial to determine treatment strategies. Several studies have examined the effectiveness of FOLFIRINOX as a first-line treatment in patients with locally advanced pancreatic cancer, but little attention has been paid to the immunologic alterations in peripheral blood caused by this chemotherapy regimen. Furthermore, the influence of the measurement type (e.g., flow cytometry and targeted gene expression) on the clinical discoveries is unknown. Therefore, we aimed to scrutinize the influence of using flow cytometry or targeted immune gene expression to study the immunological changes in blood samples of PDAC patients who were treated with a single-cycle FOLFIRINOX combined with lipegfilgrastim (FFX-Lipeg). Whole-blood samples from 44 PDAC patients were collected at two time points: before the first FOLFIRINOX cycle and 14 days after the first cycle. EDTA blood tubes were used for multiplex flow cytometry analyses to quantify 18 immune cell populations and for complete blood count tests as the standard clinical routine. The flow cytometry data were analyzed with FlowJo software. In addition, Tempus blood tubes were used to isolate RNA and measure 1230 immune-related genes using NanoString Technology®. Data quality control, normalization, and analysis were performed using nSolver™ software and the Advanced Analysis module. FFX-Lipeg treatment increased the number of neutrophils and monocytes, as shown by flow cytometry and complete blood count in concordance with elevated gene expression measured via targeted gene expression profiling analysis. Interestingly, flow cytometry analysis showed an increase in the number of B and T cells after treatment, while targeted gene expression analysis showed a decrease in B and T cell-specific gene expression. Targeted gene expression complements flow cytometry analysis to provide a comprehensive understanding of the effects of FFX-Lipeg. Flow cytometry and targeted gene expression showed increases in neutrophils and monocytes after FFX-Lipeg. The number of lymphocytes is increased after treatment; nevertheless, their cell-specific gene expression levels are downregulated. This highlights that different techniques influence clinical discoveries. Therefore, it is important to carefully select the measurement technique used to study the effect of a treatment.

Comparison of Hb A1c Quantification in the Presence of Hemoglobin Variants of an HPLC Assay with an Enzymatic Assay.

In this study, we evaluated the impact of hemoglobin (Hb) variants on the performance of the Abbott Alinity c and Bio-Rad Variant II Turbo 2.0 HPLC Hb A1c assays. The analytical performance of the Abbott Alinity c Hb A1c (enzymatic) assay was compared to the Bio-Rad Variant II Turbo 2.0 HPLC method using leftover whole blood EDTA samples with and without the presence of a hemoglobin variant. Assay precision was determined from an analysis of controls. Bias was estimated from analysis of a set of 40 samples analyzed by a Tosoh G8 HPLC instrument at the University of Missouri Diabetes Diagnostic Laboratory, an NGSP Secondary Reference Laboratory. Between-day precision was excellent for both methods (<3%). Bias met NGSP criteria of ±5% to target value. Correlation between the Alinity and Bio-Rad methods was good for patient samples without a hemoglobinopathy (y = 1.028x - 0.38, standard error of the estimate (SEE) = 0.16, n = 36, mean bias = -0.22). A total of 700 hemoglobin variant samples were evaluated on the 2 methods. Of the hemoglobin variants, 640/700 gave results on both methods: hemoglobin (Hb) S trait (n = 452), C trait (n = 131), D trait (n = 23), E trait (n = 26), and a mixture of other hemoglobinopathies (n = 8) including beta thalassemia, high hemoglobin F, transfused Hb SC, transfused Hb SD, and transfused Hb SS, or unknown variant. There was good agreement for the 640 Hb variants between the methods with a range of mean differences of -0.10 to +0.06 depending on the variant, but more variability (SEE 0.25 to 0.39). Sixty samples did not have paired results. To our knowledge, this study was the largest investigation of the effect of hemoglobinopathies on the Abbott Alinity c Hb A1c assay. Analytical performance varied depending on the specific hemoglobin variant trait when compared to the Bio-Rad Variant II Turbo 2.0 HPLC method.

Stability of PEth 16:0/18:1, 16:0/18:2, 16:0/20:4, 18:0/18:1, 18:0/18:2, and 18:1/18:1 in authentic whole blood samples (at room temperature).

Phosphatidylethanol (PEth) is a direct alcohol biomarker to monitor individuals' drinking behavior that has gained recognition in clinical and forensic settings. The increasing application of the marker makes investigation of the preanalytical handling necessary, and analyte stability deserves major attention. This study was conducted to investigate the change of six PEth homologues' concentration, stored in authentic samples of EDTA blood over a course of 30 days at room temperature (n= 62). The stability criterion of concentration being ±15% of the original concentration was fulfilled at mean for 10, 3, 2, 5, 2, and 7 days for PEth 16:0/18:1, 16:0/18:2, 16:0/20:4, 18:0/18:1, 18:0/18:2, and 18:1/18:1, respectively. Regarding all homologues, there were samples in which concentration had declined by >15% or by more than the critical difference on day 1. Overall, calculated concentration declines were very inhomogeneous, with inter-sample differences of 43%-73% after 30 days. PEth 16:0/18:2, 16:0/20:4, and 18:0/18:2 declined to a greater extent than PEth 16:0/18:1. Blood alcohol concentration was measured >0.1‰ in 25 samples. Three of the six samples that exceeded 115% of initial concentrations were positive for blood alcohol. The study results add to the previously reported information on PEth stability and firstly look at six homologues in comparison. Due to the high scatter of stability among the samples and the observed poor stabilities in some, it can be concluded that transportation and storage times, especially if cooling cannot be provided, must be kept short. If analyzing from dried blood, spotting should preferably be conducted at the site of sampling.

Anaplasma phagocytophilum Ecotype Analysis in Cattle from Great Britain.

Anaplasma phagocytophilum (A. phagocytophilum) is the aetiological agent of tick-borne fever in cattle and sheep, and granulocytic anaplasmosis in human and dogs. Livestock, companion animal and human infections with A. phagocytophilum have been reported globally. Across England and Wales, two isolates (called ecotypes) have been reported in ticks. This study examined A. phagocytophilum isolates present in livestock and wildlife in Great Britain (GB), with a particular focus on cattle. Clinical submissions (EDTA blood) from cattle (n = 21) and sheep (n = 3) were received by APHA for tick-borne disease testing and the animals were confirmed to be infected with A. phagocytophilum using a PCR targeting the Msp2 gene. Further submissions from roe deer (n = 2), red deer (n = 2) and Ixodes ricinus ticks (n = 22) were also shown to be infected with A. phagocytophilum. Subsequent analysis using a nested PCR targeting the groEL gene and sequencing confirmed the presence of ecotype I in cattle, sheep, red deer and Ixodes ricinus, and ecotype II in roe deer and I. ricinus removed from deer carcasses. Despite the presence of two ecotypes, widely distributed in ticks from England and Wales, only ecotype I was detected in cattle in this study.