Citrate Dextrose Research Articles

Effectiveness of oral calcium to prevent citrate-related toxicity during peripheral blood hematopoietic stem cell collection – An experience from a tertiary care center

Abstract: BACKGROUND: Apheresis procedures use citrate as an anticoagulant to ensure the fluidity of extracorporeal blood. Citrate-related toxicities are common adverse effects in peripheral blood stem cell (PBSC) collections as they are lengthy procedures resulting in prolonged citrate exposure. Intravenous (IV) calcium administration during therapeutic apheresis procedures is commonly used to reduce the citrate-related toxicity in adults and pediatric population. Very few literature on the use of oral calcium for the prevention of citrate toxicity during PBSC collection are present. OBJECTIVE: The objective of this study was to evaluate the effect of prophylactic oral calcium in reducing citrate-related toxicities during PBSC collections. MATERIALS AND METHODS: A prospective cross-sectional study was carried out at a tertiary care hospital of Southern India. All participants received calcium carbonate – 1 g before and at each 100 ml of collection. Preprocedure, interim, and postprocedure serum calcium were measured. All procedures were done using Spectra Optia. The ACD (acid-citrate-dextrose) rate were between 1:8 to 1:14 and were adjusted for inlet draw rate. The total blood volume processed between 2.5 to 3 based on body weight. Reduction in calcium levels and adverse reactions due to hypocalcemia were observed. Data were analyzed using SPSS 23 software. RESULTS: The majority of the procedures were undisturbed and tolerable to oral calcium. A total of 97.3% of the study subjects did not experience any symptoms of hypocalcemia. Only 2.7% (One autologous and one allogenic donor) had symptoms attributable to hypocalcemia though they had preprocedure calcium >9 mg/dl. Pre- and postprocedure calcium were compared using a paired t-test which was statistically significant. CONCLUSION: Prophylactic oral calcium in PBSC collection is an effective and easy way to the prevention of citrate-related adverse effects without affecting technical performance and product efficacy.

Operator impact assessment on qualitative and quantitative parameters of canine platelet-rich plasma.

Platelet-rich plasma (PRP) has emerged as a cornerstone in veterinary regenerative medicine. The present study evaluated the impact of the operator on the qualitative and quantitative features of non-activated PRP derived from canine whole blood. Blood was collected in anticoagulant acid citrate dextrose from twelve healthy adult dogs and PRP was prepared according to the double-spin method. Both operators followed an identical protocol and utilized the same equipment for PRP preparation from the pooled blood samples. The resulting PRP underwent characterization, classification and coding based on minimum reporting standards. The consistency and internal reliability of different parameters were also assessed using the intraclass correlation coefficient and Cronbach's alpha values. Variables such as white blood cell (WBC) concentration, relative WBC composition and mean platelet volume (MPV) showed poor reliability, and WBC concentration and MPV also had unacceptable internalconsistency. Significant differences were observed in several qualitative and quantitative parameters of the prepared PRP, highlighting the influence of the operator even when the same protocol and equipment were used. Our study has direct implications to regenerative medicine, reinforcing the urgency to set minimum requirements for reporting PRP in research studies.

Blood collection tube and anticoagulant influence on SARS-CoV-2 antibody and avidity levels

SARS-CoV-2 serology plays a crucial role in assessing COVID-19 vaccine immunogenicity and antibody responses to SARS-CoV-2 infection. Tube type and anticoagulant may influence serology results. Thus, understanding the influence of these variables in test results is key.We evaluated the influence of serum collection tube type and anticoagulant on anti-SARS-CoV-2 spike antibody levels detected by enzyme-linked immunosorbent assays (ELISAs) and Luminex multiplex assays (11-plex) in serum and plasma samples. Anti-spike IgG avidity was also evaluated in both sample types.No significant differences were found between serology assay results using different blood (serum) collection tube types. However, significantly lower antibody concentrations (p < 0.05) were observed in tubes with the anticoagulants sodium citrate and acid citrate dextrose (ACD) in the ELISA and Multiplex assays (n = 29), compared to expected concentrations. These differences mostly disappeared after adjusting for the dilution factor caused by the anticoagulant volume, indicating that anticoagulant does not significantly impact the assay results, while anticoagulant volume does.There was a significant difference (p < 0.05) in IgG avidity (M) of plasma samples (p < 0.05) compared to serum, but anticoagulant type had no effect.Overall, these findings indicate that the choice of collection tube may introduce subtle variations in assay results if the volumes of anticoagulants are not taken into consideration. Additionally, differences between serum and anticoagulant-treated plasma matrices were observed in avidity ELISAs, indicating that these samples are not interchangeable for these assays; a finding that requires further investigation.

Multivariate anomaly detection models enhance identification of errors in routine clinical chemistry testing.

Conventional autoverification rules evaluate analytes independently, potentially missing unusual patterns of results indicative of errors such as serum contamination by collection tube additives. This study assessed whether multivariate anomaly detection algorithms could enhance the detection of such errors. Multivariate Gaussian, k-nearest neighbours (KNN) distance, and one-class support vector machine (SVM) anomaly detection models, along with conventional limit checks, were developed using a training dataset of 127,451 electrolyte, urea, and creatinine (EUC) results, with a 5 % flagging rate targeted for all approaches. The models were compared with limit checks for their ability to detect atypical EUC results from samples spiked with additives from collection tubes: EDTA, fluoride, sodium citrate, or acid citrate dextrose (n=200 per contaminant). The study additionally assessed the ability of the models to identify 127,449 single-analyte errors, a potential weakness of multivariate models. The KNN distance and SVM models outperformed limit checks for detecting all contaminants (p-values <0.05). The multivariate Gaussian model did not surpass limit checks for detecting EDTA contamination but was superior for detecting the other additives. All models surpassed limit checks for identifying single-analyte errors, with the KNN distance model demonstrating the highest overall sensitivity. Multivariate anomaly detection models, particularly the KNN distance model, were superior to the conventional approach for detecting serum contamination and single-analyte errors. Developing multivariate approaches to autoverification is warranted to optimise error detection and improve patient safety.

Recognition of HLA-DPB1 alleles and their associated HLA haplotypes in 55 randomized unrelated Taiwanese individuals.

Here, we report the distribution of HLA-DPB1 alleles studied in a cohort of 55 randomly collected blood samples from unrelated Taiwanese individuals and the deduced most likely HLA haplotypes associated with the defined DPB1 alleles in the cohort. Our aim is to reveal the unprecedented data on the distribution of HLA-DPB1 alleles in the Taiwanese population and to find out the most probable HLA haplotypes associated with the HLA-DPB1 alleles detected. The material for this study was blood samples, preserved in K2EDTA and/or acid citrate dextrose anticoagulants. The blood donors were voluntary individuals of Tzu Chi Bone Marrow Donor Registry, Tzu Chi Stem Cells Center, Hualien Tzu Chi Hospital. Sequence-based typing of the Sanger's sequencing method was performed for the HLA allelic typing. To discern the HLA-DPB1 alleles, exons 2 and 3 of the HLA-DPB1 locus were sequenced. Target exon sequence amplifications were achieved by polymerase chain reaction, and the resulting amplicons were sequenced by BigDye Terminator Cycle Sequencing Ready Reaction Kit according to the manufacturer's protocols. In the total number of 55 randomized unrelated Taiwanese individuals studied, we detected 11 different HLA-DPB1 alleles. DPB1*05:01 (44.54%) was the allele with the highest frequency observed and the next highest frequency allele found was DPB1*02:01 (17.27%), while DPB1*38:01 (0.90%) and DPB1*700:01N (0.90%) ranked the least observed DPB1 alleles. Our findings in this study may be useful in researches reinforcing the comprehensive understanding on the distribution of DPB1 alleles and their associated HLA haplotypes and their clinical applications in Taiwanese.

Effect of type of anticoagulant, transportation time, and glucose in the culture media on neutrophil viability and function test results in dairy cattle.

In dairy cattle research, in vitro assessment of innate immune function is commonly evaluated by flow cytometry via the quantitative analysis of circulating polymorphonuclear leukocytes (PMN) functionalities specifically focusing on the capacities for phagocytosis (PC) and oxidative burst (OB). Variations in these PMN functions, however, may not only be influenced by the health status of the animals but also by technical, non-animal related factors. Our objectives were to assess the PMN viability, PC and OB capacities from blood samples collected in tubes coated with different anticoagulants (acid citrate dextrose (ACD) and ethylenediaminetetraacetic acid (EDTA)) and stored for 0, 3, 6, 9, and 12 h at 4°C (to mimic transportation timeframe). Furthermore, we evaluated the PMN functionalities (PC and OB) in samples incubated in culture medium with glucose (7.2 mM) versus no glucose. Over five replicates, coccygeal blood samples were collected from three nulliparous Holstein heifers (5 ACD and 5 EDTA per heifer) and allocated in a refrigerated container (4°C) for 0, 3, 6, 9, and 12 h. At each time point, PMN were isolated using gradient centrifugation. Immunolabeled PMN (CH138A) were subjected to a tricolor fluorescent staining to evaluate their viability (viable, apoptotic, and necrotic PMN). Phagocytosis and OB were assessed by incubating PMN with fluorescent beads and by phorbol 12-myristate 13-acetate stimulation, respectively. The effects of anticoagulant type, storage time, and presence of glucose in the culture medium on PMN viability and function parameters were fitted in mixed linear regression models. The proportion of viable PMN at 0 h was similar for ACD and EDTA (92 ± 4.6% and 93 ± 4.6%, respectively) but it decreased to 78 ± 4.6% for ACD and 79 ± 4.6% for EDTA after 6 h of storage. The proportion of viable PMN was not different between ACD and EDTA at any time point. The proportion of PMN that engulfed beads (PC percentage) and the PC median fluorescence intensity (MFI) reached their highest value after 3 h of storage compared with the other time points. However, the anticoagulant type (ACD versus EDTA) and the presence of glucose in the culture medium did not influence these PC parameters. Oxidative burst MFI was higher in PMN incubated in glucose-supplemented culture medium versus no glucose. We demonstrated that technical factors interfere with the evaluation of PMN viability and functionality, which can potentially lead to bias in the findings of a research hypothesis. To conclude, the present study showed that the optimal timeframe for performing PMN function analyses is within 3 hours after blood sampling. Furthermore, the presence of 7.2 mM glucose in the culture medium, a common concentration in formulation of cell culture medium, increases the in vitro OB capacity, potentially masking any impairments in in vivo PMN dysfunctionality.

Association of HLA-C*07:359 with HLA-A, -B, and -DRB1 alleles in Taiwanese.

It is thought that Taiwanese indigenous people were the "first people" to populate Taiwan (Formosa) having been there for over 5000 years, preceding the Dutch colonization (from 1624 to 1662) and Spanish colonization (from 1626 to 1642). Taiwan's indigenes, represented by Austronesian language speakers, currently constitute approximately 2% of the total population in Taiwan. It is unknown whether they evolved from Taiwan's Paleolithic or Neolithic cultures, arrived during or after the Neolithic period from China or Southeast Asia or both. HLA studies on the Taiwanese indigenous population have found several intriguing genetic information showing one or two relatively frequently observed alleles and a small number of relatively less frequently observed ones. We report here a relatively frequently observed HLA-C*07:359 allele in the Taiwanese indigenous population, its linkage with HLA-B*39:01, and its probable associated HLA haplotype in two Taiwanese indigenous families. HLA-C*07:359 is a rarely observed allele in the HLA-C locus in the world populations. The objective of this study is to report the allele HLA-C*07:359 that is more frequently found in the Taiwanese population, especially in the Taiwanese indigenous people, to demonstrate that it has a close linkage with HLA-B*39:01 allele in the HLA-B locus and to show the plausible deduced HLA-A-C-B-DRB1-DQB1 haplotypes in association with HLA-C*07:359 in two families of Taiwanese indigenous unrelated individuals. The samples were peripheral whole blood, with dipotassium ethylenediaminetetraacetic acid and/or acid citrate dextrose anticoagulation additives. The sequence-based typing method was employed to confirm the low incidence of the allele of HLA-C*07:359 observed in Taiwanese. Polymerase chain reaction was carried out to amplify exons 2, 3, and 4 of the HLA-A,-B,-C,-DRB1 and-DQB1 loci with group-specific primer sets. Amplicons were sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit in both directions according to the manufacturer's protocol. C*07:359 is an uncommon allele in the HLA-C locus in the world general population, according to our literature review. However, in this study, it is observed in the general Taiwanese population (frequency 0.41%), especially in the Taiwanese indigenous people at a frequency of 0.23%. In addition, we deduced two probable HLA haplotypes in association with C*07:359 in two indigenous families: A*24:02-C*07:359-B*39:01-DRB1*04:36 and A*24:02-C*07:359-B*39:01-DRB1*04:04. The two deduced HLA haplotypes associated with the uncommon C*07:359 allele that we report here are valuable for HLA tissue typing laboratories for reference purposes and for stem cell transplantation donor search coordinators to determine the likelihood of finding compatible donors in unrelated bone marrow donor registries for patients bearing the uncommon HLA allele. Since C*07:359 was found mostly in the Taiwanese indigenous population, we think the allele and its haplotypes we report here are important in population and anthropological studies.

A study on beneficial impact of the use of medium-molecular-weight hydroxyethyl starch in granulocyte apheresis using continuous-flow cell separator Spectra Optia: A retrospective single-center study at a tertiary care oncology center.

Granulocyte transfusion is one of the best therapeutic modalities in prolonged neutropenic patients with severe bacterial/fungal infections. Granulocyte harvest using conventional acid citrate dextrose (ACD) anticoagulant (ACD-A) by apheresis is not satisfactory in comparison to the use of hydroxyethyl starch (HES), but the latter is associated with various adverse events, especially with high-molecular-weight HES. This study aimed to assess the beneficial impact of the use of medium-molecular-weight (MMW)-HES and trisodium citrate combination over ACD-A in granulocyte apheresis when using Spectra Optia. This was a retrospective study comparing granulocyte harvest results with the use of ACD or HES and trisodium citrate combination. All the donors in both the groups received single 600 μg of granulocyte colony-stimulating factor subcutaneous injection followed by 8 mg of dexamethasone tablet 10-12 h and omnacortil 60 mg orally 3 h before harvest. A number of adverse incidents, if any, were observed and noted. Donor/procedure parameters were compared using Mann-Whitney U-test/unpaired t-test. Granulocyte yield (mean: 3.29 × 1010/unit vs. 4.5 × 1010/unit in the ACD and HES groups, respectively, P ≤ 0.0001) was significantly better in the HES group. The collection efficiency was also better in the HES group (mean: 15.86% vs. 26.70% in the ACD and HES groups, respectively, P ≤ 0.0001) in the ACD and HES groups, respectively. There was no significant adverse event noted in any of these two groups. In our study, granulocytes with optimum yield can be easily harvested with Spectra Optia cell separator using 6% HES (MMW) and trisodium citrate combination with standard 12-h interval gap between mobilization and harvest. This strategy can also have no or minimal extra cost burden to patients.

PSII-6 Changes in Galectin Gene Expression and Secretion in Response to Gum Arabica Drench in Goats

Abstract Acacia senegal is part of the natural diet of goats. Gum Arabica (GA) is a water-soluble complex polysaccharide antimicrobial, anti-inflammatory prebiotic, derived from Acacia Senegal. Its mechanism of action has not been defined. Galectins are secreted sugar-binding animal proteins involved in the regulation of innate and adaptive immunity. They function in microbe recognition and as regulators of inflammation. This study evaluated the effect of GA on Galectin transcription. Clinically healthy Boer X Spanish goats (n = 20) were randomly assigned to two groups of 10. Goats in the treatment group received 10 mL of GA in water daily for six weeks, controls received sterile water. Blood was collected weekly from the jugular vein using acid citrate dextrose as an anticoagulant. Subsequently, the blood was centrifuged to isolate the cells and plasma. Total RNA isolation was isolated from the cell pellet using Trizol (Sigma-Aldrich). The RNA concentration (ng/μL) and purity was assessed using a Nanodrop Spectrophotometer (Thermo Scientific Inc). Galectin-1, -3 and -9 transcription was conducted using goat specific primers and housekeeping genes for real-time PCR. Fold changes were calculated using the Livak method. The Log2FC values for Galectin 1, 3, and 9 expressions were 12.47, 0.29, and 2.22 respectively. Thus, GA had varying effects on structurally distinct galectins. Galectin 1 gene (Lgals1) is an important modulator of the immune response and was highly induced. Changes in Galectin transcription can impact microbial recognition and immune activation for goat health. Further analyses are needed to evaluate the functional effects of GA for application as a prebiotic and immune modulator lain goat health.

PSV-25 Effect of Garlic on Galectin 3 in Goats

Abstract The objective of this study was to determine the effect of garlic extracts on expression of Galectin 3 in goats. Inflammatory diseases such as mastitis and gastrointestinal parasitic diseases cause significant problems for goat production. Galectins (Gal) are a family of highly conserved mammalian carbohydrate-binding proteins that can regulate inflammation. Galectin-3 is the only member of the lectin family with a chimeric structure. It is a potential therapeutic target to regulate the activity of inflammatory cells. Garlic extracts are natural anti-inflammatory agents that are being evaluated for use in goat management. Twelve adult female clinically healthy Boer X Spanish goats were divided into 2 groups. The treatment group (n = 6) was drenched with a 10 mL garlic (Garlic barrier) and the control group (n = 6) was given sterile water for 30 days. Blood was collected weekly from the jugular vein using acid citrate dextrose as an anticoagulant. Blood was centrifuged to separate cells and plasma. Galectin 3 secretion in plasma was determined using goat specific commercial ELISA kits (My-BioSource). Total RNA was isolated from the cell pellet using Trizol and it was then converted into cDNA. Primers specific for goat Gal 3 specific and the housekeeping genes RPLP0 and UCHL5 (Eurofins Genomics) were used in Real-time PCR to evaluate the gene expression. Fold change was calculated using the Livak method. All variables were assessed using SAS Statistical analysis software version 9.4 (SAS Institute) at P &amp;lt; 0.05. Treatment had an effect over time and a significant increase (P &amp;lt; 0.0141) was observed in the GAL- 3 concentrations. Gal-3 was expressed in greater amounts in treatment group animals compared with the control group with a fold change of 1.15. Garlic may be used to modulate Gal 3 expression and secretion in goat blood. It is important to consider the impact of garlic on Gal 3 modulation. Studies needed to define possible impacts on immune function and goat health.

Assessment of an automated Cytomegalovirus nucleic acid amplification test using clinical plasma, bronchoalveolar lavage, and tissue specimens.

Cytomegalovirus (CMV) causes significant morbidity and mortality in immunocompromised patients, particularly transplant recipients. Quantitation of CMV DNA in peripheral blood is used to monitor prophylactic and pre-emptive approaches to prevent CMV disease, whereas CMV DNA testing of non-plasma specimens may aid in the diagnosis of end-organ disease. The analytical performance of the FDA-approved Aptima CMV Quant Assay was evaluated using reference CMV (SeraCare) diluted in defibrinated human plasma, as well as negative bronchoalveolar lavage fluid and tissue. Agreement was determined using 100 clinical acid-citrate-dextrose (ACD) plasma specimens, 77 bronchoalveolar lavage (BAL) fluids, and 101 tissues previously tested using artus CMV qPCR. Aptima CMV lower limit of detection (LLOD) was 169IU/mL for ACD plasma, 100IU/mL for BAL, and 50IU/mL for tissue. Positive percent agreement (PPA) was 100.0% (50/50; 95% CI: 92.9% - 100.0%) and negative percent agreement (NPA) was 94.0% (47/50; 95% CI: 83.5% - 98.8%) for ACD plasma. Bland-Altman analysis revealed a bias of 0.20log10 IU/mL (Aptima - artus) with 95% limits of agreement of -0.53 to 0.93. For BAL fluids, PPA was 70.0% (14/20; 95% CI: 45.7% - 88.1%) and NPA was 82.4% (43/51; 95% CI: 69.1% - 91.6%). For tissues, PPA was 90.0% (45/50; 95% CI: 78.2% - 96.7%) and NPA was 94.0% (47/50; 95% CI: 83.5% - 98.8%). The Aptima CMV Quant Assay demonstrates high analytical sensitivity and good overall agreement using clinical plasma and tissue specimens.

Blood Biomarkers Discriminate Cerebral Amyloid Status and Cognitive Diagnosis when Collected with ACD-A Anticoagulant.

The development of biomarkers that are easy to collect, process, and store is a major goal of research on current Alzheimer's Disease (AD) and underlies the growing interest in plasma biomarkers. Biomarkers with these qualities will improve diagnosis and allow for better monitoring of therapeutic interventions. However, blood collection strategies have historically differed between studies. We examined the ability of various ultrasensitive plasma biomarkers to predict cerebral amyloid status in cognitively unimpaired individuals when collected using acid citrate dextrose (ACD). We then examined the ability of these biomarkers to predict cognitive impairment independent of amyloid status. Using a cross-sectional study design, we measured amyloid beta 42/40 ratio, pTau-181, neurofilament-light, and glial fibrillary acidic protein using the Quanterix Simoa® HD-X platform. To evaluate the discriminative accuracy of these biomarkers in determining cerebral amyloid status, we used both banked plasma and 18F-AV45 PET cerebral amyloid neuroimaging data from 140 cognitively unimpaired participants. We further examined their ability to discriminate cognitive status by leveraging data from 42 cognitively impaired older adults. This study is the first, as per our knowledge, to examine these specific tests using plasma collected using acid citrate dextrose (ACD), as well as the relationship with amyloid PET status. Plasma AB42/40 had the highest AUC (0.833, 95% C.I. 0.767-0.899) at a cut-point of 0.0706 for discriminating between the two cerebral amyloid groups (sensitivity 76%, specificity 78.5%). Plasma NFL at a cut-point of 20.58pg/mL had the highest AUC (0.908, 95% CI 0.851- 0.966) for discriminating cognitive impairment (sensitivity 84.8%, specificity 89.9%). The addition of age and apolipoprotein e4 status did not improve the discriminative accuracy of these biomarkers. Our results suggest that the Aβ42/40 ratio is useful in discriminating clinician-rated elevated cerebral amyloid status and that NFL is useful for discriminating cognitive impairment status. These findings reinforce the growing body of evidence regarding the general utility of these biomarkers and extend their utility to plasma collected in a non-traditional anticoagulant.

Prevalence of baseline hypocalcemia and symptomatic hypocalcemia during leukapheresis.

Symptoms of hypocalcemia are reported in up to 50% of patients undergoing leukapheresis procedures. There is no set standard of practice for administering calcium supplementation in the prevention or treatment of hypocalcemia symptoms. The goal of this descriptive, retrospective study was to determine the prevalence of baseline hypocalcemia and symptomatic hypocalcemia during leukapheresis with acid citrate dextrose solution A and to identify patient characteristics associated with symptomatic hypocalcemia. Three percent of patients were found to have hypocalcemia before leukapheresis with 35% experiencing hypocalcemia symptoms during leukapheresis. Older age, higher albumin levels, and longer procedure time were associated with increased risk of hypocalcemia symptoms.

Plasma versus serum for extracellular vesicle (EV) isolation: A duel for reproducibility and accuracy for CNS-originating EVs biomarker analysis.

Blood-derived extracellular vesicles (EVs) are a popular source of biomarkers for central nervous system (CNS) diseases, but inconsistencies in isolation and analysis hinder their clinical translation. This review summarizes recent studies that investigate the impact of different anticoagulated plasma and serum on the yield, purity, and molecular content of EVs. Specifically, the studies compare ethylenediaminetetraacetic acid (EDTA), citrate, heparin plasma, and serum and highlight the risk of contamination from platelet-derived EVs. Here, I offer practical guidelines for standardizing EV isolation and analysis, recommending the use of plasma anticoagulated with acid-citrate-dextrose (ACD) or citrate followed by EDTA and heparin, subgroup analyses for samples from different biobank repositories, and avoiding serum and plasma-to-serum transformation. Other factors like illness, age, gender, meal timing, exercise, circadian timing, and arm pressure during blood draw can alter EV signatures. Yet, how these variables interact with different anticoagulated plasma or serum samples is unclear, necessitating further research. Furthermore, whether the changes are dependent on the isolation or quantification methodology remains an area of investigation. Importantly, the perspective emphasizes the need for consistency in experimental methodologies to improve the reproducibility and clinical applicability of CNS-originating EV biomarker studies. The proposed guidelines, along with ongoing efforts to standardize blood sample handling and collection, may facilitate the development of more reliable and informative CNS-originating EV biomarkers for diagnosis, prognosis, and treatment monitoring of CNS diseases.

Hypercoagulability in Pediatric Autologous Hematopoietic Progenitor Cell Collection – Is it the Time to Reconsider Screening? – A Case Report

Peripheral blood hematopoietic progenitor cell harvest by apheresis in pediatric patients, a double-edged sword, has advantages and disadvantages over conventional bone marrow transplantation. The effect of granulocyte colony-stimulating factor (G-CSF) mobilization on hemostasis in literature is very scarce. A 3-year-old female child with metastatic neuroblastoma planned for autologous transplant. Following G-CSF mobilization (75 mg; 4 days), the procedure was performed with all default parameters in Spectra Optia with an acid citrate dextrose (ACD) flow rate 1:12, assuming 3-total blood volume (TBV) to be processed with 150 ml of expected collection with target yield of 6 × 106 CD34 cells/kg. Despite normal coagulation laboratory parameters, it was abandoned for reasons such as low-flow alarms and thrombi in the kit tubings. In the subsequent procedures, the ACD to whole blood ratio was decreased to 1:16, and used bolus of heparin was followed by continuous heparin infusion throughout the procedure. Despite using heparin anticoagulation, a long thrombus was aspirated at needleless port. The procedure was completed with continuous low-flow alarms. A cumulative dose of 5.67 × 106 CD34 cells/kg was achieved over four procedures, but the cause of hypercoagulability was unknown. All the coagulation parameters were within normal limits before and after the procedure. The exact cause for hypercoagulability remains elusive. There are no studies or case reports in the pediatric population on hypercoagulability following apheresis procedure after G-CSF mobilization, but evidence is available for adults, with lack of explanation. It can also be due to low draw flow from the patient side or embolization of thrombi developed in the anticoagulant unprimed site in the kit tubings. More extensive studies are required to explain the mechanism of hypercoagulability during apheresis procedures in the pediatric population.

Estimation of serum calcium levels in apheresis platelet donors.

Apheresis is practiced widely to collect single donor platelets (SDPs). This procedure utilizes an anticoagulant acid citrate dextrose to prevent clotting of blood in the extracorporeal circuit which chelates divalent ions like calcium. This alters the calcium homeostasis resulting in hypocalcemia causing acute adverse events. The study aimed to know the calcium homeostasis in apheresis platelet donors. This cross-sectional study was conducted from January 2020 to December 2020 in the department of transfusion medicine. The sample size was 50. Donors who walk in for voluntary SDP donation were selected. Total and ionized calcium, pH, and serum albumin for all the donors at baseline and ionic calcium at the end of the procedure and 30 min after the procedure were measured. According to statistical analysis of the ionic calcium level at pre procedure, immediate post procedure and 30 minutes post procedure, there was decrease in the value immediate post procedure and values returned to baseline within 30 minutes. The levels of pH change were analyzed. On comparing the preprocedure and immediate postprocedure values, there was a significant lowering of pH value from the baseline (P = 0.5), indicating acute lowering of pH immediate postprocedure. Hence, most of the citrate metabolism can be achieved within 30 min after completion of the apheresis procedure. SDP collection is essentially a safe procedure with minimal adverse effects. Toxicity of citrate is not much pronounced. Recovery of calcium levels is within 30 min of completion of plateletpheresis.

104 Exposure to Environmental Pollutant GenX Chemical Modulates Innate and Adaptive Immunity Genes in Cow Neutrophils

Abstract GenX is an environmental contaminant with wide industrial use and found in drinking water. These chemicals have high bioaccumulation and cause adverse health effects in animals and humans. Previous study has shown that pre GenX perfluoroalkyl/polyfluoroalkyl substances (PFAS) and GenX affect immune cells but there is limited study on their impact on innate immune cells (neutrophils) in livestock, such as cows. This study evaluated the effect of GenX exposure on innate and adaptive immune response gene transcription in bovine neutrophils ex vivo. Whole blood was collected using Acid citrate dextrose as a coagulant from cows (n = 5). Neutrophils were isolated using differential centrifugation and hypotonic lysis of red cells. Isolated neutrophils were treated with GenX (100 ng), or untreated (control group) for hour at 37oC, 5%CO2 and 85% relative humidity. After treatment, total RNA was extracted using Trizol reagent, reverse transcribed to cDNA, and quantitative PCR was performed using the innate and adaptive immunity RT2 profiler array (Qiagen) with 84 genes. The qPCR data were analyzed using Livak’s method to calculate fold change (FC) in gene expression between GenX-treated and control neutrophils, and FC &amp;gt;2, (p &amp;lt; 0.05) was considered significant. Normalization of data was performed with GAPDH and Beta Actin as an internal control. Out of the 84 genes tested, 78 were expressed, 25 were upregulated and4 were downregulated, in response to GenX exposure. Exposure to GenX upregulated the expression of TLR8 (FC = 24.75), NOD2 (FC = 7.65), STAT1 (FC = 49.35), HLA-A (FC = 14.07), and NKB1A (FC = 16.52). Treatment with GenX decreased the expression TICAM1 (FC = -2.30), CD86 (FC = -2.27) and RAG1 (FC = -2.50). These results indicate that GenX exposure can activate and modulate the expression of innate and adaptive immune response genes in cow neutrophils. STAT1 is involved in transcriptional regulation and activation of TLR7/8 shifts neutrophil function from phagocytosis to NETosis. Thus, the mechanism of action of GenX on identified gene targets and their function and possible animal health consequences warrants further study.

DETERMINATION OF THE OPTIMUM PLATELET-RICH PLASMA METHOD FOR THE ESSENTIAL AMOUNT OF PLATELETS

Objective: Platelet-rich plasma (PRP) is the cellular plasma component containing a higher platelet concentration obtained by centrifugation of autologous peripheral blood. PRP has an auxiliary role in regenerative medicine treatment. Current treatment protocols for tissue repair strategies may be globally insufficient. PRP treatment via Platelet-released growth factors (GF) supports inflammation, proliferation, and wound healing. PRP studies have progressed and have proposed different formulations in recent years. Although various methods and devices are used, there are few and different standardized protocols to obtain PRP with the highest platelet concentration. Although anticoagulants containing citrate are primarily preferred in PRP production To provide effective platelet concentration in PRP, we noticed differences in the literature regarding PRP tube volumes, anticoagulant-citrate, and blood amounts. We aimed to obtain the most appropriate platelet concentration in PRP treatment by using different concentration ratios of Acid Citrate dextrose (ACD) solution A and different blood amounts and introducing a new treatment method to the literature. Methods: The blood of 30 individuals aged between 30 and 75 was taken into 10cc laboratory tubes, and 15cc and 18cc hourglass-shaped PRP tubes containing 8% ACD-solution A. Blood fractions were separated by centrifugation, buffy coat area was collected, and count platelet amount by Hematology Analyzer. Results: The platelet amount per µl was statistically significantly higher in 18cc blood compared to 15cc and 10cc. Platelet amounts obtained from 15 and 18cc peripheral blood with 8% ACD-solution A were above the effective treatment amount recommended in the literature. Conclusion: It was observed that the amount of used blood volume was correlated with the amount of obtained platelets.

Relationship between clinician rated amyloid status and traditional AD biomarkers in cognitively healthy older adults

AbstractBackgroundAccumulating evidence suggests that Alzheimer’s Disease (AD) plasma biomarkers aid in screening for at‐risk individuals, tracking disease progression, and monitoring response to interventions. Plasma amyloid‐β (Aβ) and phosphorylated tau (pTau) have shown promise in predicting cerebral deposition of Aβ, while neurofilament light chain (NfL) serves as a more global marker of neurodegeneration. However, blood collection protocols often differ, and it is important to confirm the relationship between blood biomarker values and established neuroimaging markers for AD neuropathology.MethodHere, we examined which blood biomarkers could predict clinician rating of cerebral amyloid status. We leveraged available clinical, cognitive, and neuroimaging data for 140 cognitively healthy older adults (65+, mean 72 ± 6.4) who participated in the Alzheimer’s Prevention Program (APP) study and had blood specimens available. Participants were assessed by a trained clinician and neuropsychometric test battery to exclude the presence of MCI or dementia syndromes. Cerebral amyloid burden was measured by Florbetapir (18F) positron emission tomography and images were rated by three trained clinicians as “elevated” or “non‐elevated”. Biomarkers were measured in plasma collected in acid citrate dextrose (ACD) tubes using a Quanterix Simoa® HD‐X platform, and included NfL, Aβ42, Aβ40, and p‐tau181.ResultPlasma Aβ42/40 ratio was significantly lower in the “elevated” group than in the “non‐elevated” group (p &lt; 0.001), while NfL (p =0.008) and p‐tau 181 (p = 0.001) were both significantly higher. Aβ42/40 ratio was able to discriminate the rating groups in a ROC analysis with an AUC of 0.835, significantly outperforming p‐tau 181 (AUC = 0.640) and NfL (AUC = 0.572).ConclusionDespite collecting samples with ACD rather than the traditional EDTA as an anti‐coagulant, expected group differences were observed. Our results suggest that Aβ42/40 ratio is useful in discriminating clinician‐rated elevated cerebral amyloid status, which has important implications regarding disclosure of blood biomarker results from different collection protocols in the context of AD risk.

Effects of Storage Temperature and Duration on Flow Cytometry Analysis of Bone Marrow Aspirate Samples

Abstract Introduction/Objective Multiparameter flow cytometry immunophenotyping is a powerful and indispensable tool in the diagnosis of hematologic malignancies, but its accuracy and precision are vulnerable to pre-analytical interferences. Acid-citrate-dextrose (ACD) is one of three main anticoagulants used for marrow collection. It offers better preservation and is commonly used for apheresis stem cell collection. However, ACD may alter the pH of short-draw samples. It is unclear how prolonged storage in ACD affects the results of flow cytometry testing. In this pre-analytical study, we characterized the effects of temperature and storage duration in bone marrow aspirate collected in ACD. Methods/Case Report Five bone marrow specimens were collected in ACD and stored at 4°C or room temperature (RT), respectively. Aliquots of specimens were tested at 24, 48, and 72 hours. A flow cytometry panel including CD19, CD33, CD34, CD38, CD45, CD138, and 7-AAD was used to identify lymphocytes, monocytes, granulocytes, myeloid blasts, hematogones, and plasma cells. Absolute cell count, percentage of leukocytes, percentage of membrane- compromised cells (7-AAD bright), and mean-fluorescent-intensities were measured for each subset. The results of each storage condition were compared to the results on the day of sample collection. Results (if a Case Study enter NA) We found that granulocytes and plasma cells are most vulnerable to delayed testing, especially when stored at RT. Myeloid blasts, hematogones, monocytes, and lymphocytes were largely stable within 48 hours at 4°C or RT, but their relative representation in total leukocytes was affected by the diminished granulocytes. Variations in surface marker expression of CD45, CD38, and CD138 in the plasma cell subset were also noticed. This effect was less profound at 4°C than RT. Conclusion We conclude that delayed testing of specimens collected in ACD within 72 hours does not significantly impact the results of flow cytometry testing for the commonly studied subsets, and 4°C may be a more robust and preferable method for storage.