Blood Donor Samples Research Articles

Naturally Occurring Anti-D in a Male Blood Donor, Unresolved Mystery.

In accordance with numerous national and international criteria, screening donated blood for abnormal antibodies against red cell antigens is crucial for patient safety. Antibodies can be formed by three mechanisms: by immune responses, naturally occurring, or passively acquired. In accordance with departmental policy, ICT was performed on a 30-yearold male blood donor's sample in addition to ABO Rh grouping. He tested A Rh Dnegative and had a positive ICT (+ 2). Anti-D was identified by using the antibody screening and identification panel. Retesting revealed similar results, so we called the donor. We were unable to find a source for the Rh isoimmunization during the course of his history. On further work-up, we found that antibodies reacted only in the antiglobulin phase with no reaction in the saline phase, suggesting an IgG type of antibody. Also, the anti-D titers (IgG) of the donor were up to 1:32. All potential sources of Rh isoimmunization have been ruled out for a male donor. Therefore, we assume that this donor has naturally occurring anti-D, perhaps created by certain environmental factors, bacteria, or components of the COVID vaccine that have cross-reactivity with the D antigen. This instance emphasizes how crucial the ICT test on the donor blood is. In our case, all potential causes of alloimmunization were ruled out, leaving only exposure to antigens with antigenic similarity to D antigens as a viable explanation.

Performance evaluation of the Access anti-HBc Total assay on the DxI 9000 Access Immunoassay Analyzer

This study evaluated the diagnostic and analytical performances of the Access anti-HBc Total assay on the DxI 9000 Access Immunoassay System (Beckman Coulter Inc.). The multicenter study involved both prospective and retrospective sample collection from non-selected blood donors, hospitalized patients, or presumed anti-HBc Total positive individuals. Fresh/previously-frozen samples were tested with the Access and comparator assays to determine concordance; discrepant samples were tested with a second CE-marked assay. Among the 5983 non-selected fresh blood donor samples deemed anti-HBc Total negative, clinical specificity of the Access assay was 99.58% (95%CI: 99.38–99.72%). Clinical specificity was 99.27% (97.37–99.80%) among 273 anti-HBc Total negative hospitalized patient samples. Clinical sensitivity on 450 anti-HBc Total positive samples was 99.78% (98.75–99.96%). Evaluation in seroconversion panels revealed an average 1.4-day earlier detection versus a comparator assay. The Access assay demonstrated excellent clinical and analytical performances comparable to existing CE-marked anti-HBc Total assays. NCT04904835.

In vitro evaluation of a new viscoelastometry-based point-of-care analyzer.

The VCM is a point-of-care analyzer using a new viscoelastometry technique for rapid assessment of hemostasis on fresh whole blood. Its characteristics would make it suitable for use in austere environments. The purpose of this study was to evaluate the VCM in terms of repeatability, reproducibility and interanalyzer correlation, reference values in our population, correlation with standard coagulation assays and platelet count, correlation with the TEG5000 analyzer and resistance to stress conditions mimicking an austere environment. Repeatability, reproducibility, and interanalyzer correlation were performed on quality control samples (n = 10). Reference values were determined from blood donor samples (n = 60). Correlations with standard biological assays were assessed from ICU patients (n = 30) and blood donors (n = 60) samples. Correlation with the TEG5000 was assessed from blood donor samples. Evaluation of vibration resistance was performed on blood donor (n = 5) and quality control (n = 5) samples. The CVs for repeatability and reproducibility ranged from 0% to 11%. Interanalyzer correlation found correlation coefficients (r2) ranging from 0.927 to 0.997. Our reference values were consistent with those provided by the manufacturer. No robust correlation was found with conventional coagulation tests. The correlation with the TEG5000 was excellent with r2 ranging from 0.75 to 0.92. Resistance to stress conditions was excellent. The VCM analyzer is a reliable, easy-to-use instrument that correlates well with the TEG5000. Despite some logistical constraints, the results suggest that it can be used in austere environments. Further studies are required before its implementation.

Clinical significance of viral markers testing by ELISA and Individual Donation Nucleic Acid Testing (ID-NAT) for blood screening in blood bank: Single center study in Egypt

Prevention of transfusion-transmitted viral infections and insurance of safe blood transfusion are the main goals of all blood banks worldwide. Despite the high sensitivity and specificity of currently used enzyme linked immunosorbent assay (ELISA) for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) testing, viral transmission could still occur during the window period. Introducing viral individual donation nucleic acid testing (ID-NAT) can greatly decrease such risk providing an additional layer in securing blood transfusion. We aimed to assess the clinical significance of viral markers testing by ELISA and ID-NAT for blood screening in the Blood Bank of Suez Canal University Hospital. We studied all donations (2132) collected during a two-months period. Blood donor samples were screened by ELISA and ID-NAT tests for HBV, HCV, and HIV. Serological testing results for HCV by ELISA revealed 2,122 (99.5 %) negative donations compared to 2,131 (99.95 %) negative donations by ID-NAT testing. Of the positive ELISA samples, only one was NAT positive. For HBV ELISA testing, 2,115 (99.2 %) donations were negative, also by ID-NAT testing 2,115 (99.2 %) donations were HBV DNA negative. Out of the negative ELISA samples, two samples were ID-NAT reactive donors which were missed by serology assay being in the window period. HIV ELISA testing revealed negative 2,130 (99.9 %) donations while ID-NAT testing showed 2,131 (99.95 %) negative donations and one positive donation. In conclusion, this is the first study carried out in the Suez Canal and Sinai region, Egypt to assess the importance of ID-NAT implementation. The introduction of ID-NAT in blood banks is an effective method for increasing safety of the blood transfusion.

Comparison of results obtained using clot-fibrinolysis waveform analysis and global fibrinolysis capacity assay with rotational thromboelastography

Global fibrinolysis assays detect the fibrinolysis time of clot dissolution using tissue-type plasminogen activator (tPA). Two such assays, clot-fibrinolysis waveform analysis (CFWA) and global fibrinolysis capacity (GFC) assay, were recently developed. These were compared with rotational thromboelastography (ROTEM). Healthy donor blood samples were divided into four groups based on tPA-spiked concentrations: 0, 100, 500, and 1000 ng/mL. CFWA and GFC fibrinolysis times, including 4.1 µg/mL and 100 ng/mL tPA in the assays, were determined, denoted as CFWA-Lys and GFC-Lys, respectively. Statistical differences were recognized between tPA concentrations of 0 and 500/1000 ng/mL for CFWA-Lys, and 0 and 100/500/1000 ng/mL for GFC-Lys. The correlation coefficients with lysis onset time (LOT) of extrinsic pathway evaluation and intrinsic pathway evaluation in ROTEM were statistically significant at 0.610 and 0.590 for CFWA-Lys, and 0.939 and 0.928 for GFC-Lys, respectively (p-values < 0.0001 for all correlations). Both assays showed significant correlations with ROTEM; however, the GFC assay proved to have better agreement with ROTEM compared with the CFWA assay. These assays have the potential to reflect a hyperfibrinolysis status with high tPA concentrations.

SARS-CoV-2 seroprevalence in Nova Scotia blood donors.

SARS-CoV-2 seroprevalence monitors cumulative infection rates irrespective of case testing protocols. We aimed to describe Nova Scotia blood donor seroprevalence in relation to public health policy and reported data over the course of the COVID-19 pandemic (May 2020 to August 2022). Monthly random Nova Scotia blood donation samples (24,258 in total) were tested for SARS-CoV-2 infection antibodies (anti-nucleocapsid) from May 2020 to August 2022, and vaccination antibodies (anti-spike) from January 2021 to August 2022. Multivariable logistic regression for infection antibodies and vaccination antibodies separately with month, age, sex, and racialization identified independent predictors. The provincial nucleic acid amplification test (NAAT)-positive case rate over the pandemic was calculated from publicly available data. Anti-N seroprevalence was 3.8% in January 2022, increasing to 50.8% in August 2022. The general population COVID-19 case rate was 3.5% in January 2022, increasing to 12.5% in August 2022. The percentage of NAAT-positive samples in public health laboratories increased from 1% in November 2021 to a peak of 30.7% in April 2022 with decreasing numbers of tests performed. Higher proportions of younger donors as well as Black, Indigenous, and racialized blood donors were more likely to have infection antibodies (p < 0.01). Vaccination antibodies increased to 100% over 2021, initially in older donors (60+ years), and followed by progressively younger age groups. SARS-CoV-2 infection rates were relatively low in Nova Scotia until the more contagious Omicron variant dominated, after which about half of Nova Scotia donors had been infected despite most adults being vaccinated (although severity was much lower in vaccinated individuals). Most COVID-19 cases were detected by NAAT until Omicron arrived. When NAAT testing priorities focused on high-risk individuals, infection rates were better reflected by seroprevalence.

Abstract 7489: Peripheral blood liquid biopsy for the screening of lung cancer

Abstract Lung cancer is the second most common type of cancer in both men and women and the leading cause of cancer-related mortality. Unfortunately, most patients have advanced stages (stage III or IV) at the time of diagnosis and the only U.S. Preventive Services Task Force-recommended screening test, Low Dose CT scan (LDCT), has a high false-positive rate. Therefore, there is an urgent need for the early detection of lung cancer with better discrimination between individuals with lung cancer and those without. In this study, we demonstrate the capability of a non-enrichment-based liquid biopsy workflow, the High Definition Single Cell Assay (HDSCA) to be used as a complementary tool to the current screening methodology. The HDSCA workflow can detect circulating cells of epithelial, mesenchymal, endothelial, and hematological origin, as well as acellular events such as oncosomes. This allows the investigation of the broad spectrum of circulating rare events for the potential identification of biomarkers associated with lung cancer. We investigated 52 peripheral blood samples collected from the second annual screening round of Manchester’s 'Lung Health Check' pilot of a community-based lung cancer screening cohort. Of these, 7 were subsequently diagnosed with non-small cell lung cancer, 2 with small cell lung cancer, and 43 were deemed at high risk of developing lung cancer. We additionally investigated 60 peripheral blood samples from individuals with no known pathology to use as controls. Within the screening cohort, a number of single rare analytes were found to have statistically significant differences between the lung cancer, high-risk cohort, and normal blood donor samples, which can potentially be biomarkers used to differentiate the cohorts. We also built a multi-analyte patient-level classification model utilizing the rare cellular and acellular events that stratified lung cancer patients from the normal donor individuals and high-risk cohort with high accuracy. Our results demonstrate the potential of liquid biopsy as a complementary screening approach for lung cancer. Citation Format: Jiyoun Seo, Stephanie N. Shishido, Jeremy Mason, Carmen Ruiz Velasco, Andrew Chen, Dominic Rothwell, Phil Crosbie, James Hicks, Caroline Dive, Peter Kuhn. Peripheral blood liquid biopsy for the screening of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7489.

Analyzing Blood Cells of High-Risk Myelodysplastic Syndrome Patients Using Interferometric Phase Microscopy and Fluorescent Flow Cytometry.

Myelodysplastic syndromes (MDSs) are a group of potentially deadly diseases that affect the morphology and function of neutrophils. Rapid diagnosis of MDS is crucial for the initiation of treatment that can vastly improve disease outcome. In this work, we present a new approach for detecting morphological differences between neutrophils isolated from blood samples of high-risk MDS patients and blood bank donors (BBDs). Using fluorescent flow cytometry, neutrophils were stained with 2',7'-dichlorofluorescin diacetate (DCF), which reacts with reactive oxygen species (ROS), and Hoechst, which binds to DNA. We observed that BBDs possessed two cell clusters (designated H and L), whereas MDS patients possessed a single cluster (L). Later, we used FACS to sort the H and the L cells and used interferometric phase microscopy (IPM) to image the cells without utilizing cell staining. IPM images showed that H cells are characterized by low optical path delay (OPD) in the nucleus relative to the cytoplasm, especially in cell vesicles containing ROS, whereas L cells are characterized by low OPD in the cytoplasm relative to the nucleus and no ROS-containing vesicles. Moreover, L cells present a higher average OPD and dry mass compared to H cells. When examining neutrophils from MDS patients and BBDs by IPM during flow, we identified ~20% of cells as H cells in BBDs in contrast to ~4% in MDS patients. These results indicate that IPM can be utilized for the diagnosis of complex hematological pathologies such as MDS.

Blood donation screening for hepatitis B virus core antibodies: The importance of confirmatory testing and initial implication for rare blood donor groups.

Exclusion of blood donors with hepatitis B virus (HBV) core antibodies (anti-HBc) prevents transfusion-transmitted HBV infection but can lead to significant donor loss. As isolated anti-HBc positivity does not always indicate true past HBV infection, we have investigated the effectiveness of confirmatory anti-HBc testing and the representation of rare blood groups in anti-HBc-positive donors. Three hundred ninety-seven HBV surface antigen-negative and anti-HBc initially reactive blood donor samples were tested by five different anti-HBc assays. Eighty percentage of samples reactive in Architect anti-HBc assay were positive by the Murex assay and anti-HBc neutralization. Eleven out of 397 samples showed discordant results in supplementary testing from the Murex confirmatory test result, and five remained undetermined following extensive serological testing. Thirty-eight percentage of anti-HBc-positive donors identified as minority ethnic groups compared with 11% representation in anti-HBc-negative donors (p < 0.0001); the frequency of the Ro blood group in anti-HBc-positive donors was 18 times higher in non-white ethnic groups. Using two anti-HBc assays effectively enabled the identification of HBV-exposed and potentially infectious donors, their deferral and potential clinical follow-up. However, the exclusion of confirmed anti-HBc-positive donors will still impact the supply of rare blood such as Ro.

Clinical trial and performance evaluation of the Wantai HBsAg (CMIA) diagnostic kit for screening blood donors in China

In China, according to the ‘Technical Operating Procedures for Blood Stations (2019 Edition),’ blood stations are authorized to utilize Chemiluminescence Immunoassay (CLIA) to detect pathogen markers linked with transfusion-transmissible infections. However, currently, there is no approved CLIA reagent for the screening of blood-borne diseases in China, specifically for the detection of Hepatitis B surface antigen. The objective of this research project is to conduct a comprehensive evaluation of the performance of the Wantai Chemiluminescent Microparticle Hepatitis B surface antigen reagent. This study evaluates the performance of the Wantai Chemiluminescent Microparticle Immunoassay (CMIA) on the Wan200 + analyzer in screening for Hepatitis B Surface Antigen (HBsAg) in blood samples. The clinical trial component of this evaluation is included as part of the documentation submitted to the National Medical Products Administration (NMPA) of China for the approval of blood screening reagents. The evaluation plan of this study encompasses two main components: clinical trials and performance assessment. We adopted a controlled trial design, utilizing the WanTai Chemiluminescent Microparticle Immunoassay (CMIA) on the Wan200 + analyzer and the Enzyme-Linked Immunosorbent Assay (ELISA) to screen for Hepatitis B Surface Antigen (HBsAg) in routine blood donor samples and reference serum panel samples. To ensure the accuracy of the screening, we additionally employed Abbott's ELISA reagents and HBV DNA for validation. The assessment primarily focused on key performance indicators such as sensitivity, specificity, and analytical sensitivity. Moreover, this clinical trial data has been included as part of the submission to China's National Medical Products Administration (NMPA). In the clinical trials of this study, a total of 10,470 blood donor samples underwent simultaneous testing using both CMIA and ELISA methods. Across two clinical trials, there was remarkable concordance between CMIA and the two ELISA reagents, with Kappa values exceeding 0.82. Among the 269 samples that were double-reactive in the enzyme immunoassay (ELISA) tests, CMIA exhibited a 100% reactivity detection rate. However, CMIA produced 14 and 6 false-positive results in the respective clinical trials, resulting in specificities of 99.73% and 99.89%. In contrast, the specificities for Wantai ELISA and Xin Chuang ELISA were both greater than 99.94%.When testing samples in the gray zone serum plates, CMIA's detection limit significantly exceeded that of the two ELISA assays. CMIA had a detection cutoff of 0.05 IU/mL, while the two ELISA reagents had cutoffs of 0.1 IU/mL and 0.09 IU/mL, respectively. CMIA's detection limits for the adr and adw subtypes were 0.05 IU/mL, and for the ay subtype, it was 0.1 U/mL. The detection limit for 10 HBV mutant samples was 0.5 U/mL. In 165 cases where ELISA tested negative but HBV DNA tested positive, CMIA detected 5 HBsAg-positive samples. This study evaluated the performance of the Wantai CMIA in screening for HBsAg among blood donors. The results demonstrate outstanding performance of CMIA in both clinical trials and performance assessments, detecting all true positive samples with a sensitivity of 100%. It exhibits excellent concordance with the two ELISA assays. Of particular note is its superiority in early detection of HBsAg in the screening of early-stage hepatitis B infections, reducing the window period compared to ELISA. CMIA achieves a specificity exceeding 99.73% for negative blood donors, aligning with the European Union's standards for blood screening assay specificity. In summary, Wantai's CMIA displays high sensitivity and specificity in blood donor screening, making it suitable for screening blood donors in China.

Evaluation and experience from routine use of chemiluminescence assays for serological screening of blood and plasma donations on the Alinity s system and the Alinity i system, two new fully-automated immunoassay systems in Poland

In Poland, independent evaluations under the auspices of the Institute of Hematology and Transfusion Medicine (IHTM) are mandated for any new device, assay, systems for screening samples from whole blood and plasma donors prior to implementation by Blood Transfusion Center (BTC). In last 5 years, two new systems were introduced to the market by Abbott GmbH, namely the Alinity s and the Alinity i. The evaluations performed for these two systems included the assessment of sensitivity, specificity and precision for each of the four mandatory serological screening markers in Poland: Hepatitis B Surface Antigen (HBsAg), Hepatitis C virus antibodies (Anti-HCV), HIV antibodies (anti-HIV) and Syphilis antibodies (anti-Treponema pallidum, anti-TP). Sensitivity was assessed by testing seroconversion panels, HBsAg international reference standard, well characterized local samples, and dilution panels. Specificity was assessed by testing routine donor samples. The results from Alinity i assays were compared to the results from Abbott ARCHITECT i2000SR and Ortho VITROS 3600 assays, while the results from Alinity s assays were compared to the results of ARCHITECT i2000SR assays. The evaluation of the Alinity s and Alinity i assays for sensitivity (100 %), specificity (99,92–100 %) and precision generated results that were as good as or better than generated by routinely used systems, were within acceptance criteria, and met all requirements for screening blood donor samples in accordance with Polish regulations. The specificity of the assays in routine use by BTCs, analyzed after approximately 150,000 donations on both systems, was comparable to the specificity observed during the evaluations at IHTM.

First Report of Phenotype Prevalence of Five Major Rh Blood Group Antigens in the Blood Donor Population of Peshawar, Pakistan

Objective: The current study investigated the prevalence of five major Rh blood group antigens in the Peshawar region of Khyber Pakhtunkhwa province, Pakistan.&#x0D; Methodology: This was a cross-sectional study, conducted at the Peshawar Regional Blood Centre from February to April 2023. A total of 13,148 blood donor samples were analyzed for Rh D, C, c, E, and e antigens using monoclonal reagents. The frequencies were calculated by direct counting on a spreadsheet programme and the Statistical Package for Social Sciences (SPSS for Windows version 25.0. Armonk, NY, USA) was used to conduct a meta-analysis for comparison of results with national and international studies.&#x0D; Results: The findings of the current study revealed that 92.17% of donors were RhD positive. The e antigen showed the highest frequency of 98.31%, followed by D antigen and the C antigen (92.17% and 78.37%, respectively). Comparative analyses with earlier Pakistani studies and international data indicated significant variations in antigen frequencies, potentially attributed to the diverse ethnic population and geography of the region.&#x0D; Conclusion: The findings emphasize the importance of local antigen frequency data for efficient blood transfusion strategies, especially for thalassaemia patients requiring frequent transfusions.&#x0D; Key words: Rhesus, Blood Group, Phenotype

Evaluation of the efficacy and safety of lyophilized plasma

Objective. To assess the hemostatic potential, preclinical safety and in vitro efficacy of lyophilized plasma in patients and blood donors.Materials and methods. The study of acute toxicity was carried out on outbred mice of both sexes (n=120), pyrogenicity – on rabbits of both sexes (n=30), abnormal toxicity – on mice (n=25) and guinea pigs (n=6). Histopathological studies were carried out on preparations of five fragments of the middle part of the mouse tail after intravenous administration of the maximum dose (4500 mg/kg) lyophilized plasma (LP), physiological solution, fresh frozen plasma (FFP) and pathogen-reduced FFP (PRFFP) using the Intercept system. The proand anticoagulant activity was investigated by clotting and chromogenic method to evaluate the hemostatic potential of PL. The effect on hemostasis was studied in vitro by thromboelastometry when FFP and LP were added to blood samples of patients (n = 23) and donors (n = 20). Results. Acute toxicity study showed the absence of death and clinical signs of intoxication in all series. On the 14th day, body weight gain was observed in animals of all series. Regardless of the used LP dosage, no development of effusion and adhesions in the thoracic and abdominal cavities, as well as macroscopically detectable pathologic changes of the main organs were observed in animals. The analysis of the relative weight of internal organs of animals did not reveal statistically significant differences in experimental and control series relative to the values of similar indicators in intact animals. The studied hemostatic potential showed that LP was not inferior to PRFFP in the content of coagulation factors of the blood system. Evaluation of the effect of LP application in vitro in female patients showed statistically significant correction of CT (clotting time) index, which indicates the effectiveness of its application for replenishment of blood coagulation factors (BCF).Conclusion. Preclinical animal studies of LP have proven its safety and efficacy compared to FFP.

Seroprevalence of Transmisive Diseases in Healthy Blood Donors: a Five-year Experience in University Clinical Center Tuzla.

The safety of blood products is the most important task of transfusion medicine. Transfusion-transmitted diseases represent a serious public health problem throughout the world, although their percentage of transmission is minimal. Each blood donation is individually tested with serological and molecular tests for the presence of hepatitis B and C viruses, human immunodeficiency virus (HIV), and Treponema Pallidum, regardless of the number of blood donations. To determine the seroprevalence of blood-borne diseases in healthy blood donors at the University Clinical Center Tuzla, to analyze the possible causes of transmission of these diseases, and to compare with the results of research in countries in the region and beyond. The research was conducted retrospectively in the period January 1, 2018. until 31.12.2022. years. Samples of blood donors were tested for hepatitis B surface antigen (HBsAg), antibodies to human immunodeficiency virus (HIV1/2), antibodies to hepatitis C and syphilis, using the fourth generation ELISA method. The highest frequency of transmisive disease markers was reactivity to HBsAg with a total of 63.7% of cases and a prevalence of 0.13%, and the lowest for HIV with a total of 1.4% and a prevalence of 0.003%. Bosnia and Herzegovina has a very low seroprevalence but there is still a risk of disease transmission in the population.

Prevalence and identification of irregular erythrocyte antibodies in voluntary blood donors in the territory of southeast Serbia

Background/Aim. The purpose of tests for transmittable diseases, as well as determining ABO group, Rh typing, and the presence of clinically significant erythrocyte antibodies, is to enable the appropriate selection of blood/blood components for transfusion, to prevent or minimize adverse effects of transfusion, and to identify donors whose units of blood/components are not suitable for transfusion. The aim of the study was to evaluate the presence of irregular erythrocyte antibodies as well as to determine the specificity and origin of these antibodies in voluntary donors. Methods. This prospective observational study, conducted from January until July 2023, included samples of voluntary blood donors (BDs) for antibody screening in the population of voluntary BDs in the territory of Southeast Serbia. A total of 23,082 samples from healthy BD were examined through this period. Results. The overall prevalence of irregular anti-erythrocyte antibodies was 0.1125%, with anti-K, anti-D, and anti-E being the most frequently identified alloantibodies. Out of a total of 26 donors with positive screening results, 11 (42.32%) had alloantibodies, and 15 (57.68%) had antibodies of undetermined specificity. A positive antibody test was observed more frequently in male donors, and the age range of 51 years and above accounted for the majority of positive cases. Conclusion. Immunohematological testing, including antibody screening in voluntary BDs, is important for safe blood transfusion and is of great clinical importance. Likewise, it reduces the risk of complications due to incompatible transfusions.

Ultrasensitive detection of circulating tumor cells in clinical blood samples by a three-dimensional network nanovehicle-based aptasensor platform

A burgeoning topic in the early diagnosis and treatment of cancer is the sensitive and precise quantification of circulating tumor cells (CTCs). However, due to the extremely low CTC concentrations in the blood, traditional enrichment and identification methods for CTCs are prone to false negative results. Here, to specifically identify and improve the sensitivity of CTC detection in the blood, we designed a novel fluorometric turn-on aptasensor platform based on a three-dimensional network nanovehicle (TDNN) modified with a PEG derivative and AS1411 aptamer specifically targeting the nucleolin protein. The limit of detection (LOD) of the aptasensor platform was as low as 2 cells per mL, which was remarkably better than that of the single nanoparticle ASS1. Notably, the novel aptasensor platform showed excellent specificity and sensitivity as well as negligible fluorescent background for quantitative and visualization detection of CTCs in peripheral blood samples without enrichment, centrifugation and washing steps. More importantly, the number of CTCs identified by TDNN was significantly higher in metastatic patients than in nonmetastatic patients, while CTCs were not detected in the blood samples of healthy donors. In conclusion, the aptasensor platform provides an available and promising tool for CTC identification and cancer metastasis prediction in clinical blood samples.

Lymphocyte Cell Expressions and SARS-COV-2 Antibody Titers in Healthy Donors’ Blood at an Indonesian Blood Transfusion Center (UTD PMI Surabaya)

Highlights: 1. There was no relationship between total lymphocyte count and antibody titers against SARS-CoV-2.2. This original research report offers information on the several blood test result after getting COVID-19vaccines and provides data that the public may use as scientific evidence to the further research. Abstract SARS-CoV-2 is a virus that infects the respiratory tract and attacks the respiratory mucosa and epithelial cells.Lymphocytes are a subset of agranulocyte leukocytes that have a role in the immune response to pathogenicmicroorganisms' attacks. The number of lymphocytes will increase if a virus causes an infection. An antibody is aprotective protein produced by the body's immune system in response to the presence of an antigen. Antibodies bind and inactivate foreign substances so that the replication of pathogens can be prevented and destroyed. This mechanism can be expressed through the total number of lymphocyte cells. The objective of this study was to define the relationship between the number of lymphocytes and antibody titers against SARS-CoV-2. The type of research used was analytic observational with a cross-sectional approach using quantitative analysis methods and a purposive sampling method for sampling. The samples used were 30 donor blood samples from UTD PMI Surabaya. This research was conducted in May 2022 at the Hematology Laboratory, Department of Medical Laboratory Technology, Poltekkes, Ministry of Health, Surabaya, to examine the number of lymphocyte cells using the flowcytometry method and the Immunoserology Laboratory of the Surabaya Health Laboratory Center for antibody titer examination using the ELISA (Enzyme-Linked Immuno Sorbent Assay) method. The study showed an average lymphocyte level of 2.2633 x 103/µl and an average antibody titer value expressed in OD of 0.2197. Data analysis using Spearman's rank correlation statistical test revealed p = 0.262 0.005. It showed no relationship between total lymphocyte count and antibody titers against SARS-CoV-2

Advancing Hematological Point-of-Care Diagnostics: Development and Multi-Site Evaluation of a Novel AI-Enabled Diagnostic Device for CBC Analysis with Differential

Introduction: A complete blood count (CBC) with differential is a routine blood test that provides essential health information that is fundamental for maintaining wellness. While many other routine tests can be run in Point of Care (POC) settings, a CBC with differential is largely confined to central laboratory testing environments. Truvian is in late-stage development of a fully automated device that runs a TruWellness Panel (TM) incorporating all routine tests - a CBC with 3-part differential, comprehensive metabolic panel (CMP), lipid panel, HbA1c and TSH in a single run with 300uL of whole blood. This device incorporates high-throughput cell imaging, Computer Vision (CV) and Artificial Intelligence (AI), including machine learning and deep learning, for on board CBC analysis with 3-part and 5-part differentials. Here, we summarize our development and evaluation of the AI-enabled CBC sub-panel through method comparison studies. Methods: An AI-Enabled 5-part white blood cell (WBC) differential was developed using transfer learning. WBC cells were detected through classical object detection algorithms and extracted from fluorescent images of normal donor blood samples acquired on the Truvian device. Each cell in the set was classified by hematopathologists into one of five categories - Neutrophil, Lymphocyte, Monocyte, Eosinophil, and Basophil - to train a deep learning model. The algorithm currently outputs the data in a 3-part differential format (neutrophils, lymphocytes and “others”) with refinement of the full 5-part differential in progress. The final training dataset consisted of ~2000 - 5000 cells in each class. The training set was divided into training (80%) and test (20%) datasets. Data augmentation techniques were leveraged to improve model invariance to cell orientation and reduce the risk of overfitting. Performance of each iteration of the algorithm was assessed using accuracy of the model on test data. Following incorporation of the algorithm into Truvian's comprehensive and automated blood testing platform, comparability between an external reference laboratory and Truvian's system was performed. Samples from over 150 donors were measured and analytical concordance was evaluated by Passing-Bablok regression, and bias was evaluated using the TOST equivalency test. Results: WBC differential models were improved over a two-year period to incorporate cell image refinement associated with hardware and software iterations. The final algorithm was able to accurately detect and differentiate WBCs into 3 main categories: neutrophils, lymphocytes, and “others” (monocytes, eosinophils, and basophils). Neutrophils, lymphocytes, and “others” demonstrated R correlations of 0.98, 0.96 and 0.49 respectively. Slopes of 1.0 were observed for both neutrophils and lymphocytes while “others” had a slope of 1.37. Intercepts ranged from -0.07 to 0.04 x10 3 cells/μL for the various cell classes. The RBC, WBC and platelet cell counting algorithms were able to produce excellent correlation compared to predicate devices with correlation coefficients ≥ 0.95 and slopes between 0.96 to 1.03. Conclusions: In conclusion, this novel diagnostic device represents a breakthrough in point of care diagnostics. The compact, fully automated blood diagnostic device can run a CMP, lipid panel, CBC with 3-part differential, HbA1c and TSH assays simultaneously. By incorporating Computer Vision algorithms for cell detection and AI techniques such as machine and deep learning for differentiation on board the instrument, it offers an efficient tool for accurate and precise CBC analysis with 3-part differential at the point-of-action. Further development and refinement of the differential algorithm is currently underway.

Development of a Fast and Quantitative FLT3-TKD Mutation Prototype Using Lab in a Cartridge™ Technology

Introduction Acute myeloid leukemia (AML) is a heterogenous hematologic malignancy characterized by the rapid proliferation of abnormal myeloid cells in the bone marrow and blood. Fast and reliable diagnostic tests are imperative for the advancement of targeted therapies in the treatment of AML. FLT3 (FMS-like tyrosine kinase3) is one of the most frequent genetic alterations in AML and is present in approximately one-third of newly diagnosed patients. Considering the targeted therapies, such as midostaurin and gilteritinib available for FLT3 mutated AML, accurate and timely diagnosis of FLT3 mutations is vital for guiding treatment decisions and improving patient outcomes. The current diagnostic methods to detect FLT3-TKD mutations require a series of complex steps including blood sample handling, plate-based PCR, enzyme digestion and capillary electrophoresis or sequencing which can be time-consuming and labor-intensive. We have developed and evaluated the performance of a FLT3-TKD prototype using Cepheid's automated cartridge technology that demonstrates significantly less hands-on time and faster turnaround. Objectives We have developed a novel, fast and simplified qPCR-based FLT3-TKD prototype using Cepheid's GeneXpert® automated platform for detecting 14 mutations, including 7 mutations present in codon D835 and another 7 mutations in I836. The FLT3-TKD prototype is an easy-to-use sample in/result out assay with minimal hands-on time and time to results in less than 3 hours, and without the use of restriction enzymes or capillary electrophoresis. This assay is designed for quantitative detection of FLT3-TKD mutated DNA (D835 &amp; I836) using ABL1 as an endogenous control in peripheral blood (PB) of AML patients. This assay utilizes the special chemistry, special design primers, and an optimized wild-type (WT) blocker to specifically detect SNPs and indels. With the combination of WT blocker and unique design of special design primers we were able to selectively amplify the rare mutations in the presence of abundant closely related WT fragments. Results Using this prototype, we were able to detect and quantify all 14 mutations (7 of D835 and 7 of I836) with an estimated LoD of AR 0.02 (Ratio of TKD mutation copies/ABL copies in PB background) in PB samples spiked-in with positive templates. ABL1 serves as a reference gene to normalize the WT DNA present in the background. Plasmid DNA with individual mutations was generated and spiked into the PB to assess the dynamic range of the test. We observed good alignment of dCt-AR (deltaCt-Allelic ratio) regression curve among all 14 mutations within the dynamic range tested (0.02-0.33). With pre-defined thresholds, our assay specificity has been above 95% among 20 screened EDTA donor blood samples. To further assess the sensitivity of this prototype, we also evaluated a CRISPR-CAS engineered cell line for the D835 G&amp;gt;A mutation. Using this cell line, we were successful in detecting the mutation down to AR of 0.008 in a PB background. For quantification analysis we prepared a linear panel of the most prevalent mutation D835(G&amp;gt;T) in the PB background and were able to quantify all mutations within the range of AR 0.008-to 0.83. Conclusions Here we describe the development and evaluation of an automated cartridge-based FLT3-TKD prototype that can detect 14 FLT3-TKD mutations. The cartridge prototype is preloaded with sample processing reagents and qPCR reagents with a turn-around time of less than 3 hours including offboard peripheral blood processing. When tested with contrived samples generated with either plasmid DNA or engineered cell lines spiked into blood, all 14 tested FLT3-TKD mutations were detected within a dynamic range of 0.02 to 0.33 AR with a specificity of &amp;gt; 95% in healthy donor whole blood background. With optimized primer and WT blocker design this prototype showed high sensitivity &amp; specificity. We tested a broader dynamic range for the most prevalent mutation, D835 G&amp;gt;T, and were able to detect from AR of 0.008 to 0.83 AR. Additionally, since the prototype uses the GeneXpert® platform and current universal lysate sample prep procedure, the sample can potentially be used across all Cepheid Hematology Oncology products. This prototype significantly reduces the hands-on time with future potential to provide clinicians with fast and accurate detection of FLT3 mutation status that can aid prognostic evaluation and treatment in AML patients.

A Pan-Cancer Single-Cell Transcriptomic Atlas of Natural Killer (NK) Cells Reveals Intrinsic and Extrinsic Mediators of NK Cell Anti-Tumor Functions

Background: Natural killer (NK) cells are critical mediators of anti-tumor immunity, engaging in direct cytotoxic responses and promoting pro-inflammatory immune microenvironments. The molecular mechanisms underlying these functions, however, have yet to be fully elucidated. This is due in part to a lack of understanding of the phenotypic and functional diversity of NK cells across cancer types. Here, we present a single-cell transcriptomic atlas of &amp;gt;250,000 NK cells spanning 340 patients across 17 cancer types to advance our understanding of tumor-infiltrating NK cells. Methods: We re-analyzed publicly available single-cell RNA-sequencing (scRNA-seq) datasets of primary, therapy-naïve tumors, as well as matching peripheral blood samples and healthy donor blood samples. We employed systematic computational analyses to deconvolute the phenotypic and functional diversity of tumor-infiltrating NK cells and define key regulators of NK cell functions in both a cancer type-specific and pan-cancer manner. Feature expression module scoring was used to infer phenotypic and functional characteristics of NK cell clusters in each cancer type. Transcription factor regulon and cell-cell communication prediction algorithms were applied to understand the mechanistic drivers of NK cell populations within tumors. Results: Tumor-infiltrating NK cells display distinct patterns of phenotypic and functional features across cancer types. Nonetheless, we identify consensus NK cell populations across cancers that demonstrate the utility and performance of our gene module-based phenotypic and functional annotations. Interestingly, we observe the enrichment of adaptive-like and CD56 bright NK cell populations in tumors that exhibit features of tissue residency, including the upregulation of ITGA1, ITGAE, and CD69 alongside the downregulation of FCG3RA. Notably, these tissue-resident populations display enhanced chemokine and cytokine activity compared to their recruited counterparts, suggesting the significance of tissue-resident NK cell populations in immune recruitment to tumors. We find the phenotypic composition of intra-tumoral NK cells is vastly different than that of circulating NK cells which harbor predominantly CD56 dim populations, as well as a significant proportion of adaptive-like NK cells in several cancer types. Leveraging the paired nature of several tumor and peripheral blood samples, we performed temporal comparative analyses that revealed the robust expression of KLF2 and S1PR5 in circulation which is then lost in paired tumor tissues. While KLF2 and S1PR5 have been previously implicated in the homing and migration of immune cells, their roles in the trafficking of NK cells to tumors have yet to be defined. In modeling transitions from canonical to stressed NK cell states, we not only identify well-established NK cell checkpoints ( TIM-3, LAG3, and CISH) but also identify novel putative checkpoints yet to be investigated in NK cells, such as the AP-1 family of transcription factors. Additionally, we examine extrinsic factors contributing to intra-tumoral NK cell phenotypes, detecting significant crosstalk between NK cells and immunomodulatory macrophages and CD4+ T cells mediated by galectin-9 and TGFβ interactions. Conclusions: We define the phenotypic and functional pan-cancer landscape of NK cells and uncover both intrinsic and extrinsic factors influencing their anti-tumor functions. We envision our NK cell atlas will facilitate the development of strategies to enhance NK cell-based immunotherapies against cancer.