Donor Serum Research Articles

An oriented self-assembly biosensor with built-in error-checking for precise midkine detection in cancer diagnosis and prognosis evaluation

Midkine (MDK) is a neurotrophic growth factor highly expressed during embryogenesis, currently recognized as a multifaceted factor in cancer progression and drug resistance. MDK has demonstrated greater accuracy than existing biomarkers. Serum MDK is a valuable indicator for the non-invasive early detection of tumors. It dynamically changes following surgical tumor excision and prior to recurrence, facilitating prognosis and treatment response evaluation. However, existing methods struggle to achieve the sensitivity required for clinical applications. Herein, we developed a triple-mode biosensor with oriented self-construction and built-in error-checking for rapid, sensitive, and convenient MDK detection. The sensor construction adhered to the principle of achieving oriented and strong covalent connections to ensure high sensitivity. Biosynthesized quantum dots (BQDs) were introduced to orient antibodies, enhancing the exploration of active binding sites and significantly increasing antibody-capturing ability. To further enhance sensitivity and signal amplification, Au@Pt nanorods-Ab2 (MF-Probe) were used as multifunctional probes, incorporating an error-checking mechanism to minimize false results. Detection was feasible using an electrochemical workstation, a microplate reader, and even a mobile phone. The sensor exhibited a wide linear range from 5 fg/mL to 100 ng/mL and a low limit of detection (LOD) of 1.620 fg/mL. It accurately distinguished MDK levels in the serum of healthy donors and cancer patients. Compared to existing ELISA kits, it exhibited a lower LOD and a more sensitive response to trace MDK, suggesting it is a promising tool for cancer diagnosis and prognostic evaluation.

Donor and Recipient Factors Associated with Primary Graft Dysfunction Following Lung Transplantation: A DMG Registry Analysis

Current risk-adjusted models to predict primary graft dysfunction (PGD) following lung transplantation (LTx) do not include bedside donor critical care data. Donor management goals (DMG) represent predefined critical care endpoints aimed at optimizing multi-organ donor management. Here, we sought to identify novel predictors to better understand the relationship between donor management and PGD following LTx. We used the national DMG registry to identify a cohort of LTx recipients linked to their respective donors between January 1st, 2015 and March 1st, 2023. Grade 3 PGD (PGD3) was defined according to modified ISHLT criteria. Multivariable modeling was performed to identify risk factors for the development of PGD3. A total of 2704 eligible patients were identified of which 643 (23.8%) developed PGD3. After multivariable modeling, the likelihood of PGD3 was greater with increased donor age (OR 1.06 [1.02, 1.10] per 5 year change, p=0.003), increased donor serum pH at the time of authorization (OR 1.14 [1.02, 1.25] per 0.1 increase, p=0.016), donor history of cocaine use (OR 1.34 [1.05, 1.71], p=0.020), and increased recipient central venous pressure (1.03 [1.01, 1.06], p=0.005). Recipients who received donor lungs in which the DMG for PF ratio was met had a lower likelihood of developing PGD3 (OR 0.63 [0.46, 0.86], p=0.006). This study leverages a novel detailed donor management database to identify factors associated with the development of PGD3. These factors may be used to recognize donors and recipients that may benefit from early interventions to improve short-term outcomes.

Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions.

Background/Objectives: Cripto-1 (CR1) is a plurifunctional embryonic protein required for implantation and re-expressed in the adult during wound repair, inflammation, and tumorigenesis. CR1 and its predicted CR1 pseudogene product Cripto-3/CR3 are highly homologous proteins, and given this physical attribute, commercially available antibodies cannot discriminate between CR1 and CR3. Methods: A series of mouse monoclonal antibodies [MoAbs] were developed with a high-affinity binding that can differentiate human CR1/CR3 proteins and showed no measurable cross-reactivity. Results: Using these reagents, we confirm that CR3 is a bona fide translated protein found in human tumor tissue, cancer cell lysates, and in normal/cancer patient donor sera. We also reveal that CR1 and CR3 compete for binding to signal transduction protein Nodal, glucose-regulated protein 78Da (GRP78), and activin receptor-like kinase 4 (Alk4). Our discriminatory MoAbs provide new reagents to help clarify current CR1/CR3 protein expression vagaries in the Cripto field of study, challenging established CR1 conventions. In addition, our data validate CR3 involvement in human carcinogenesis and cell signaling pathways, with potential clinical relevance in determining cancer patient prognosis and disease severity.

6651 A Traceable Clinical Free Thyroxine Assay Based on Equilibrium Dialysis ID-LC/MS/MS

Abstract Disclosure: D. Spector: None. L. Zhang: None. A. Ribera: None. A. Doty: None. C. Tse: None. O. Sugahara: None. N. Vazquez: None. U. Danilenko: None. H.W. Vesper: None. Accurate diagnosis and effective treatment of thyroid diseases rely on reliable thyroid function tests. Serum free thyroxine (FT4) immunoassays (IAs) are most commonly used for FT4 measurements in clinical practice. However, FT4 IAs have not been standardized with large variations between different assay platforms. A reference measurement procedure (RMP) for FT4 based on well-defined equilibrium dialysis (ED) of serum has been established for CDC standardization program to support the standardization of clinical assays for patient care. In the current study, we aim to develop a routine clinical FT4 assay based on ED-LC/MS/MS that is traceable to the RMP. ED-LC/MS/MS analysis was performed using a commercially available micro-ED plate. FT4 in dialysate was extracted with a 96-well C18 SPE plate. FT4 in the samples was quantitated by using LC/MS/MS in positive ion mode. The assay was calibrated with the certified primary reference material. By using RMP as a reference, we compared the analysis of 40 single donor sera between the ED-LC/MS/MS assay and FT4 IA on Roche e401platform. ED-LC/MS/MS assay could resolve T4, T3, reverse triiodothyronine (rT3), and other interferences in the samples with C18 chromatography in 4 min. The linear range of the routine FT4 assay was from 0.5-100 pg/mL covering clinically relevant FT4 concentrations including hypo-, hyperthyroid patient samples. The intra and inter-run CV% of the assay in 20 days was 3.72 and 5.25%, respectively. The effect of minor changes to the method conditions was studied. It was determined that serum/buffer ratios from 1:1 to 1:2 in the ED inserts, speeds of shaker for ED, and ED incubation time, did not influence the FT4 measurement. However, elevated ED temperature at 38°C could result in a more than 10% increase in FT4 values. A microplate format for the procedures of ED and SPE is amiable to the application of an automation system which significantly improves the throughput of the procedure. Method comparison showed good agreement between ED-LC/MS/MS assay and RMP in Deming regression analysis with a slope close to 1, while there was significant bias of IA from RMP. ED-LC/MS/MS assay was also applied for pregnancy serum samples with minimal difference on the FT4 measurement from RMP. We have developed a routine clinical FT4 assay that is traceable to RMP. The accuracy, precision, and sensitivity of the assay are suitable for high-throughput FT4 measurements in clinical laboratories and large epidemiologic studies. We demonstrate that the developed method can provide accurate FT4 measurements in patients including pregnant women. Presentation: 6/3/2024

6848 Improvement Of Free Thyroxine Measurement Quality Through The CDC Clinical Standardization Programs

Abstract Disclosure: A. Ribera: None. N. Vazquez: None. S. Knoblock: None. O. Sugahara: None. T. Buchannan: None. L. Collins: None. D. Spector: None. L. Zhang: None. U. Danilenko: None. H.W. Vesper: None. Thyroid function tests are among the most requested tests in patient care, and reliability of these measurements are crucial for appropriate diagnosis and management of thyroid disorders. In response to concerns from the clinical and research community about the quality of thyroid function tests, the CDC Clinical Standardization Programs (CSP) has created the hormone standardization (HoSt) program for free thyroxine (FT4) in partnership with the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC). As a first step, CDC CSP has completed an interlaboratory comparison study of commercial immunoassays (IA) and lab-developed tests (LDT) to determine the current agreement among assays. Participants received 40 blinded, high quality individual donor serum samples with FT4 reference values assigned by the CDC FT4 reference measurement procedure (RMP). FT4 concentrations ranged between 11-32 pmol/L. Samples were analyzed in duplicate over 2 days and results were compared to the CDC FT4 RMP. To assess the potential impact of standardizing measurements to the CDC HoSt FT4 program, reported results were normalized to the CDC RMP results (recalibrated in silico using linear regression). Mean percent bias to the reference value was evaluated pre- and post-recalibration. In addition, inter-assay agreement of classification (assignment of hypo-, eu- or hyperthyroid status according to FT4 reference intervals) were assessed pre- and post-recalibration using the assay-specific and CDC RMP reference intervals, respectively. A total of 19 methods participated, consisting of 15 IA and 4 LDT. All participants showed a negative mean bias to the RMP, with IA showing a higher magnitude of bias (average: -20.7 ± 0.9%) than LDT (average: -4.7 ± 1.1%). Of the 40 samples measured, only 53% were classified uniformly by all 19 participants. Post-recalibration, mean percent bias improved profoundly (IA average: 0.0 ± 0.8%, LDT average: 0.1 ± 1.1%), and classification agreement improved by 33% to 86% of samples with consistent classification. The CDC CSP interlaboratory comparison among FT4 assays highlights the critical need for measurement standardization to improve accuracy of FT4 measurements and increase consistency in result interpretation. This study demonstrates that it is possible to develop common FT4 reference intervals via measurement standardization, which would allow for universal evidence-based clinical guidelines to be used in patient care. Presentation: 6/3/2024

Expedient measurement of total protein in human serum and plasma via the biuret method using fiber optic probe for patient samples and certified reference materials.

The biuret method is currently recognized as a reference measurement procedure for serum/plasma total protein by the Joint Committee for Traceability in Laboratory Medicine (JCTLM). However, as the reaction involved in this method is highly time-dependent, to ensure identical measurement conditions for calibrator and samples for high accuracy, a fast and simple measurement procedure is critical to ensure the precision and trueness of this method. We measured serum/plasma total protein using a Cary 60 spectrophotometer coupled with a fiber optic probe, which was faster and simpler than the conventional cuvette method. The biuret method utilizing alkaline solutions of copper sulfate and potassium sodium tartrate was added to the sample and calibrator (NIST SRM 927e) incubated for 1h before measurement. A panel of samples consisting of pooled human serum, single donor serum, and certified reference materials (CRMs) from three sources were measured for method validation. Sixteen native patient samples were measured using the newly developed biuret method and compared against clinical analyzers. Additionally, the results of three cycles of a local External Quality Assessment (EQA) Programme submitted by participating clinical laboratories were compared against the biuret method. Our biuret method using fiber optic probe demonstrated good precision with within-day relative standard deviation (RSD) of 0.04 to 0.23% and between-day RSD of 0.58%. The deviations between the obtained values and the certified values for all three CRMs ranged from -0.38 to 1.60%, indicating good method trueness. The routine methods using clinical analyzers were also found to agree well with the developed biuret method using fiber optic probe for EQA samples and native patient samples. The biuret method using a fiber optic probe represented a convenient and reliable way of measuring serum total protein. It also demonstrated excellent precision and trueness using CRMs and patient samples, which made the method a simpler candidate reference method for serum protein measurement.

A-038 Interlaboratory Comparison of Parathyroid Hormone Analytical Results Across Clinical Assays from 8 Manufacturers - Steps Towards Standardization

Abstract Background Parathyroid hormone (PTH) is an 84 amino acid peptide produced by the parathyroid gland and is a key biomarker used for the diagnosis and treatment of Chronic Kidney Disease-Mineral and Bone Disorder, as well as hypo- and hyperparathyroidism. PTH clinical assays were previously reported to exhibit significant inter-assay measurement variability, which limits the utility of PTH as a diagnostic biomarker and can result in the misclassification of patients. Thus, there is a need for PTH clinical assay standardization to improve clinical assay measurement agreement. The Centers for Disease Control and Prevention’s Clinical Standardization Program (CDC-CSP) will launch a PTH standardization program, which is in support of and coordinated with the IFCC Committee on Bone Metabolism. In preparation, the CDC-CSP conducted an Inter-Laboratory Comparison Study to evaluate the current status of PTH inter-assay variability and to investigate potential sources of variability. Methods Eight PTH assay manufacturer’s laboratories measured PTH in 42 deidentified, fresh-frozen single donor serum samples in duplicate over 2 independent runs. Sample PTH concentrations ranged from 1.3 - 324 pg/mL, based on initial screening values. Five manufacturers submitted data for 2 different assays, resulting in PTH measurements from 13 assays total. Data were submitted to the CDC CSP for analysis. Results Assay precision was <5% for 10/13 measurement procedures (MP) assessed and 3 MPs exhibited precisions between 5 - 18%. Assay-specific PTH measurements demonstrated good linear correlation compared to the all-assay mean concentration. Interestingly, inter-assay variability increased with increasing PTH concentrations, with individual sample standard deviations increasing from 2.9 - 123, with a mean SD of 24.1 and individual sample CVs increasing from 18.9 - 44.5%, with a mean CV of 24.2%. Conclusions This study provides new insights into PTH inter-assay variability and assay performance for assays commonly used in the clinical setting. The findings of this PTH Interlaboratory Comparison Study establish a baseline for PTH inter-assay variability and will guide future PTH standardization efforts.

B-099 Validation and Clinical Implementation of Organ Procurement Serology Testing: Our Analytical and Regulatory Experience in Implementing a New Donor Testing Program

Abstract Background The Mount Sinai Hospital recently implemented a comprehensive organ donor testing program to service the clinical needs of transplant services in the New York City metro area. Conventional serology testing is not approved by the FDA for donor testing. To support this program, the MSH Chemistry Laboratory implemented and validated a comprehensive donor testing panel on the Abbott Alinity S donor testing platform. We validated donor serology testing for the following assays: HIV-1/2 (HIV), Anti-Hepatitis B Core (Anti-HBc), Hepatitis B Surface Antigen (HBsAg), Human T-lymphotropic virus I and II (HTLV-I/II), Anti-Hepatitis C (Anti-HCV), and Trypanosoma cruzi antibodies (T.Cruzi). The Alinity S System is an automated immunoassay-based analyzer FDA-approved for donor testing. The analyzer consists of two distinct sample ‘paths’ and is effectively two instruments combined in one analyzer. The analyzer is approved for use in living donor serum and plasma and in cadaveric serum. Prior to going live, we found that there was limited guidance on laboratory quality assurance programs related to donor testing. In addition to confirming assay analytical performance, we designed and implemented a comprehensive quality assurance program prior to satisfy compliance with regulatory guidelines, addressing proficiency compliance, sample TAT, and control ranges, amongst other quality indicators. Methods Inter and Intraday precision was assessed through replicate analysis of positive and negative controls, where 10 replicates of both controls were assayed on a single day, and one replicate of each control was assayed daily for 7 distinct days, results were represented as %CV. Result accuracy was assessed through split sample analysis of known serologically positive (n=20) and negative samples (n=40). Each sample was assayed on two distinct lots of reagents, each with its own unique calibration curve. Accuracy was confirmed in both living donor (n=30), and cadaveric samples (n=10). In addition, split sample analysis was performed between the Alinity S and FDA-approved donor testing assays in a reference laboratory (N=7). A carryover study was performed through the analysis of triplicate replicates of low, high, and then low controls. These analyses were performed for all 6 assays. Results Intraday precision using assay general controls afforded the following %CV: HIV: 3.1%, 6.5%; Anti-HBc: 1.3%, 2.6%; Anti-HCV: 3.3%, 11.7%; HTLV-I,II: 2.5%, 5.9%; T.Cruzi: 2.8%, 0%; HBsAg: 3.2%, 11.6%. Interday precision yielded the following in %CV: HIV: 2.9%, 10.0%; Anti-HBc: 3.0%, 5.6%; Anti-HCV: 3.3%, 3.4%; HTLV-I,II: 2.8%, 4.6%; T.Cruzi: 2.4%, 0%; HBsAg: 4.7%, 7.8%. Accuracy studies yielded 100% positive and negative correlation for all 6 assays in both living donor and cadaveric samples. In addition, there was a 100% serological correlation for samples compared to reference laboratories. Carryover studies showed no evidence of sample carryover. Conclusions The Abbott Alinity S shows acceptable assay precision on all 6 validated assays. The assays are serologically accurate in both cadaveric and living donor samples. As such, the assays and platform are acceptable for use in donor screening. We designed a unique quality assurance program including a comprehensive self-proficiency and instrument crosscheck program to ensure analyzer accuracy, as well as adherence to regulatory guidelines.

B-080 Development and Validation of a GQ1b IgG ELISA as a Diagnostic Aid for Subacute Demyelinating Polyneuropathies

Abstract Background Autoimmunity targeting GQ1b is associated with group of disorders that includes Miller-Fisher Syndrome (MFS), Bickerstaff Brainstem Encephalitis (BBE) and classic Guillain Barre Syndrome (GBS) with ophthalmoplegia. Collectively these are referred to as GQ1B-IgG related syndromes. The prevalence of GQ1b-IgG in this population of patients is high and has been reported to be >80% in well-defined clinical cohorts. Clinical phenotypic presentation is diverse and requires highly accurate antibody testing. The objective of this study was to evaluate a newly developed custom GQ1b-IgG assay, using a semi-quantitative ELISA-based method. Methods A GQ1b-IgG ELISA was developed and validated. Patient sera along with assay controls and a single-point calibrator are diluted and incubated in duplicate wells in a 96-well plate coated with GQ1b antigen. The plate is washed and incubated with anti-human-IgG secondary antibody conjugated with HRP. After a colorimetric reaction, optical densities (OD) are read. The OD of patient samples are compared against the calibrator, generating a cut-off index (COI). Samples ≥1.0 COI are considered positive. During the validation phase, analytical and clinical performance characteristics were evaluated. Studies included assessment of imprecision at multiple concentrations of GQ1b-IgG, accuracy using method comparison, analytical sensitivity and specificity, reference range, common interferences, plate drift, and clinical performance in relevant disease and control cohorts. Results Intra-assay imprecision (coefficient of variation; %CV) ranged from 1.0 - 16% across all concentrations of GQ1b-IgG. Inter-assay imprecision (%CV) ranged from 12.4 - 44.5% but was <20% at or above the assay cut-off. We found perfect qualitative agreement with an independent ELISA reference method (20 positive and 20 negative samples selected based on reference method results [reference interval <1:100]). Limit of detection (LOD) was calculated at a COI of 0.22. To assess analytical specificity, 40 samples with high concentrations of immunoglobulins (hypergammaglobulinemia [N=15], IgG M-protein [N=15] and systemic lupus erythematosus [N=10] were tested and all were negative. A reference interval study was performed on 140 healthy donor serums, all were negative. Interference studies were conducted on 2 positive and 2 negative serum samples, spiked for highly hemolytic (1000 mg/dL), lipemic (2000 mg/dL) and icteric (60 mg/dL) conditions. There was no impact on the assay even with high levels of a given interferent. Plate drift on 2 distinct patient pools tested across the plate (COI range 0.7 - 1.3) had overall plate CV’s ≤10%. ROC analysis was performed on a cohort of 278 patient samples (15 MFS cases and 263 controls [suspected demyelinating neuropathy or disease mimics]). A cut-off value of 1.0 (COI) was selected based on the Youden index. At this cut-off, clinical sensitivity for MFS was 87% while clinical specificity was 99.2%. Conclusions The observed performance of the GQ1b-IgG ELISA was acceptable for clinical laboratory implementation. A COI of ≥ 1.0 was selected for clinical use.

B-102 The Impact of Protein / Protein Interactions on Serum Free Light Chain Assay Calibration

Abstract Background Since 2001, Serum Free Light Chain measurements have become established as routine clinical laboratory tests. Katzmann (2002) published a reference interval based on 282 normal samples establishing a ratio reference range 0.26-1.65. Whilst kappa and lambda measurements are of little diagnostic value, kappa/lambda ratio gives a strong indication of monoclonal gammopathy. In recent years, it has been suggested that the ratio may have changed. Here we present investigations identifying protein/protein interactions that may account for the discrepancies between QC release and real-world data; identifying a remediation step which will allow verification of the published ratio. Methods The manufacturers QC release data was reviewed for kappa, lambda and kappa/lambda ratio for all lots from 2018-2023, and compared to published data. To mimic field observation, an accelerated stability study was performed on standard calibrators and protein/protein interactions identified using western blot. Subsequently, calibrators were depleted of alpha-1 antitrypsin (A1AT) and other serum proteins using specific affinity chromatography; accelerated stability was repeated. A calibrator of pure polyclonal free light chains (FLC) in human serum albumin was produced in the presence/absence of A1AT and additional interfering serum proteins. Results At release, the average kappa reference range was 7.2-29.5 mg/L (62 kits, 9 different US blood donor sera collections and 3852 data points) and 7.7-24 mg/L for lambda (78 kits, 9 different US blood donor sera and 4664 data points), with a kappa/lambda ratio of 0.45-1.69. There was minimal change over 5yrs to these measurements. Analysis of FLC calibrators during an accelerated stability identified interactions between kappa FLC and ubiquitous serum proteins, including A1AT. The standard calibrator activity declined by 14.9±0.1% (mean±SEM) over a 4-month accelerated period @22oC, equivalent to 16 months real time. Calibrators depleted of A1AT and specific additional serum proteins showed significantly less decline in activity 1.4±1.4%, p<0.001. When blood donor sera were analysed using the standard calibrators following 4 months at 22oC, the results showed an increase in the reported kappa concentrations compared to those generated from frozen calibrators (19.0[15.4-22.5] to 21.3[17.0-26.0] mg/L, 12.5±3.8%, p<0.001). This observation was mirrored in kappa/lambda ratio (1.3[1.1-1.5] to 1.4[1.2-1.7] mg/L, 12.5±3.8%, p<0.001) and when clinical patient samples were assessed (30.1[14.0-47.1] to 34.3[15.5-56.9] mg/L, 13.8±1.4%, p<0.001). The depleted calibrator did not report an increase in sample results. FLC+HSA calibrators, when spiked with either physiological concentrations of A1AT or other serum proteins, showed a 16.7% and 15.3% decrease in activity respectively, compared to FLC+HSA calibrators without additional proteins. Conclusions At release, US blood donor sera have different absolute kappa mg/L compared to the published reference interval, but these changes do not invalidate the published kappa/lambda ratio, in contrast to recent reports and have not changed since 2018. An unpredictable kappa/protein interaction was observed, particularly with A1AT in a time dependent fashion, which correlated to a reduction in calibrator activity. Removal of A1AT and other specific serum proteins remediated the issue, and the kappa/lambda ratio was verified irrespective of the age of the calibrator.

A-338 Assessing the Technical and Analytical Performance of Point of Care Testing Devices for Blood Lipids Measurements

Abstract Background Cardiovascular diseases (CVDs) are the leading cause of death worldwide, causing over 17 million deaths each year. According to the World Health Organization (WHO), 85% of CVD deaths are due to heart disease and strokes, and over 75% occur in low- and middle-income countries (LMICs). Early detection and treatment are key to preventing consequences of CVD. Point-of-care testing (POCT) devices require minimal laboratory infrastructure and can be used to screen hard-to-reach populations for CVD risk. These devices are increasingly used in traditional care settings including hospitals and primary healthcare, and in non-traditional settings such as pharmacies. Although POCT devices offer fast and convenient testing, the analytical performance of these devices must produce accurate and reliable results, and have technical suitability for use. Several POCT devices for total cholesterol (TC) and other blood lipids are commercially available, however, the operational and analytical performance of these devices is not fully known. Methods Eight POCT devices for blood lipids were used in this study. For the technical assessment, a checklist was developed and applied, outlining the operational functionality and environmental conditions of the devices. Some relevant technical parameters in this checklist included requirements for temperature/humidity and power/voltage, storage conditions, and assay/test time. The analytical performance was assessed for parameters, such as accuracy and imprecision, using paired whole blood and serum donor samples for TC. In addition, these analytical parameters were assessed for serum High Density Lipoprotein-cholesterol (HDL-c) and Low Density Lipoprotein-cholesterol (LDL-c) samples on seven POCT devices. All samples were representative of a clinically relevant concentration range for TC, HDL-c, and LDL-c. Accuracy was compared to reference values obtained by the CDC reference measurement procedures. Results The mean bias for TC ranged among devices from -8.17% to 11.99% for whole blood and -7.89% to 7.02% for serum, respectively. The mean imprecision for TC in whole blood ranged from 1.94% to 11.63%, and in serum from 1.72% to 12.70%. The mean bias for serum HDL-c samples ranged from -7.33% to 17.36%, and for serum LDL-c samples from -20.92% to 5.04%. The mean imprecision for serum HDL-c samples ranged from 2.39% to 24.27% and for serum LDL-c samples ranged from 3.14% to 18.33%. For three of the eight devices, about 25% of the whole blood measurements and 50% of the serum measurements were reported outside of the measurement range and not included in the assessment. Comparing these non-reports with the reference values of these samples indicate that all of the reference values for whole blood and about 80% of the reference values for serum were actually within the measurement range of the device. Conclusions The technical assessment was helpful in building screening capacity in areas with limited laboratory infrastructure. The analytical assessment indicates notable differences in accuracy and precision for lipids measured in whole blood and serum, with serum measurements being more likely within performance requirements used by CDC's standardization programs. Additional studies to assess other devices and/or analytes, and future work to assist manufacturers improve the analytical performance of POCT devices are needed.

Development and validation of a model for early survival prediction following liver transplantation based on donor and recipient characteristics

Background Circulating cytokine levels not only correlate with the progression of liver disease but also serve as indicators for the infection status of the body. Growing evidence points to the connection between donor cytokines and graft function following transplantation. This study set out to explore the clinical significance of donor cytokines in predicting liver transplantation prognosis. Methods Data from 172 deceased donor liver transplantations conducted between 2017 and 2022, with available donor serum cytokine information, were collected. The subjects were randomly divided into estimation (n = 120) and validation (n = 52) groups to establish and validate the model. The newly developed SA10 score was compared against established models EAD, MEAF, L-GrAFT7, and L-GrAFT10. Results Donor IL-10, along with donor age and recipient AST peak value within the first 7 days post-operation, was identified as an independent factor associated with recipient survival and was incorporated into the SA10 score. SA10 exhibited robust predictive capability, particularly for 1-month survival (AUC = 0.90, 95% CI = 0.84–0.96), outperforming EAD (AUC = 0.75, 95% CI = 0.60–0.90, p = 0.04) and L-GrAFT7 (AUC = 0.65, 95% CI = 0.49–0.81, p < 0.01). Comparable performance was observed between SA10, MEAF, and L-GrAFT10. Conclusion Donor IL-10 independently influences recipient survival, with the SA10 score demonstrating comparable and even superior predictive ability compared to existing models.

Cell-based in vitro hemoassay for evaluation of NLRP3-inflammasome activity

Currently, gouty arthritis is considered as a polygenic multifactorial autoinflammatory disease caused by activation of the NOD (nucleotide-binding domain) -like protein receptor 3 inflammasome NLRP3. The two cytokines IL-1β and IL-18 are considered important biomarkers of NLRP3 inflammasome activation. However, usually the concentration of IL-1β in donor sera is extremely low and found to be at the limit of detection level (1-3 pg/ml), while the concentration of circulating cytokine IL-18 in the sera of individual donors varies greatly. This results in difficulty using these biomarkers in the diagnosis of autoinflammatory diseases. We hypothesized that the patient’s blood cells which were sensitized in vivo to the presence of specific factors characteristic of autoinflammatory diseases, in particular, gouty arthritis, would produce increased amounts of the inflammasome-regulated cytokines compared to blood cells obtained from healthy donors. A comparison of the IL-18 cytokine in healthy donors and patients with gouty arthritis was carried out using 2 methods: a) by traditional analysis of the level of serum circulated IL-18 and b) by using a cell-based Hemoassay in vitro developed at the research institute “Biotech” SamGMU. The comparative analysis demonstrated the advantages of using an in vitro cell-based Hemoassay to assess the IL-18 cytokine status of patients. Serum IL-18 values varied widely and showed no significant difference between donors and patients with gouty arthritis. Using the developed cell-based Hemoassay in vitro, significant quantitative differences in the production of the inflammatory cytokine IL-18 produced by blood cells of potentially healthy donors and patients with gouty arthritis were detected. Blood cells of individual patients, sensitized in vivo with specific factors characteristic of gouty arthritis, produce increased concentrations of IL-18 in the cell growth medium in vitro compared to cells from healthy donors. Thus, the in vitro cell-based Hemoassay can be used for a more accurate assessment of the cytokine status of patients.

The role of C4d and donor specific antibodies in face and hand transplantation-a systematic review.

To date, little is known about the mechanisms of rejection in vascularized composite allotransplantation, particularly for antibody mediated rejection. Additionally, no clear guidelines exist for the diagnosis and management of antibody-mediated rejection in vascularized composite allotransplantation. A systematic review of electronic databases (Embase and PubMed) was conducted to evaluate the relationship of donor specific antibodies and C4d deposition in correlation with cellular rejection following hand and face transplantation reported by centers between 1998 and July 2023. We extracted data on serum donor specific antibodies at the time of biopsy proven rejection according to Banff classification and C4d staining of target tissues. Mann-Whitney U tests were performed to compare rejection grade between groups divided by status of C4d deposition and serum donor specific antibodies, and Fisher's Exact test was used to assess association between the two markers. This review adhered to PRISMA guidelines. A total of 26 patients (5 face, 21 hand) were identified and data on 90 acute rejection episodes with information on Banff grade, donor specific antibody status, and C4d deposition were available. Donor specific antibodies were found to be associated with higher rejection grade (p = 0.005). C4d was not found to be associated with higher rejection grade (p = 0.33). Finally, no significant association was found between concurrent status of the two markers (p = 0.23). These findings suggest that the presence of donor specifc antibodies may be associated with higher grades of acute cellular rejection following hand and face transplantation. More consistent reporting on rejection episodes is needed in order to better understand antibody-mediated rejection in vascularized composite allotransplantation.

Free radical fragmentation and oxidation in the polar part of lysophospholipids: Results of the study of blood serum of healthy donors and patients with acute surgical pathology

The interaction of reactive oxygen species with cell membrane lipids is usually considered in the context of lipid peroxidation in the nonpolar component of the membrane. In this work, for the first time, data were obtained indicating that damage to human cell membranes can occur in the polar part of lysophospholipids at the interface with the aqueous environment due to free radical fragmentation (FRF) processes. FRF products, namely 1-hexadecanoyloxyacetone (PAc) and 1-octadecanoyloxyacetone (SAc), were identified in human serum, and a GC-MS method was developed to quantify PAc and SAc.The content of FRF products in serum samples of 52 healthy donors was found to be in the range of 1.98–4.75 μmol/L. A linear regression equation, CPAc&SAc (μmol/L) = 0.51 + 0.064 × years, was derived to describe the relationship between age and content of FRF products. In 70 patients with acute surgical pathology in comparison with the control group of healthy donors, two distinct clusters with different concentration levels of FRF products were revealed. The first cluster: groups of 43 patients with various localized inflammatory-destructive lesions of hollow organ walls and bacterial translocation (septic inflammation) of abdominal cavity organs. These patients showed a 1.5–1.9-fold (p = 0.012) decrease in the total concentration of PAc and SAc in serum. In the second cluster: groups of 27 patients with ischemia-reperfusion tissue damage (aseptic inflammation), – a statistically significant increase in the concentration of FRF products was observed: in 2.2–4.0 times (p = 0.0001).The obtained data allow us to further understand the role of free-radical processes in the damage of lipid molecules. FRF products can potentially be used as markers of the degree of free-radical damage of hydroxyl containing phospholipids.

Molecular genetic characterization of hepatitis B virus in blood donors from South Vietnam

The problem of transfusion safety preventing parenteral viral hepatitis transmission remains relevant. Viral hepatitis B (HB) is the most common viral infection transmitted through transfusion procedures. One of the natural phases of a chronic viral hepatitis B (CHB) course is occult hepatitis B infection (OBI) characterized by undetectable HBsAg level (regardless of other serological marker levels) along with detected hepatic HBV DNA as well as blood viral load ranging from extremely low to undetectable. In Vietnam, prevention of transfusion-based HBV transmission is focused on donor screening; it is still based solely on HBsAg serology. As such, OBI remains a potential threat to blood transfusion safety. Assessing hepatitis B virus (HBV) DNA is a reliable preventive measure against HBV transmission from HBsAg– donors, especially in highly endemic regions. The aim of our work was HBV identification and molecular genetic characterization in blood donors from South Vietnam. The study material was presented by 500 donor serum samples. Subjects were examined for HBV markers with qualitative detection of HBsAg, HBs IgG, and HBcore IgG. Amplification and subsequent HBV sequencing were performed using nested PCR with overlapping primer pairs jointly flanking the complete HBV genome (S, P, C, X genes). Full-size HBV genome nucleotide sequences were obtained for 58 samples. Among blood donors, taking into account HBsAg+ and HBsAg– samples, HBV DNA was detected in 11.6%, including 8.6% OBI. HBV phylogenetic analysis showed genotypes B and C. Vaccine escape mutations and mutations that contribute to disease progression were identified. Current screening in Vietnam is insufficient for eliminating the risk of transfusion-transmitted HBV infection. The major risk factor is OBI. PCR testing for HBV should be considered for blood donor screening.

Prediction of Renal Graft Function 1 Year After Adult Deceased-Donor Kidney Transplantation Using Variables Available Prior to Transplantation.

BACKGROUND Kidney transplantation is still the best therapy for patients with end-stage renal disease, but the demand for donor organs persistently surpasses the supply. A prognostic model using pre-transplant data for the prediction of renal graft function would be helpful to optimize organ allocation and avoid futile transplantations. MATERIAL AND METHODS Retrospective data of 2431 patients who underwent kidney transplantation between January 01, 2000, and December 31, 2012 with subsequent ten-year clinical follow-up in our transplant center were analyzed. Of these, 1172 patients met the inclusion criteria. Multivariable regression modelling was used to develop a prognostic model for the prediction of graft function after 1 year utilizing only pre-transplant data. The final model was assessed with the area under the receiver operating characteristic (AUROC) curve. RESULTS Donor age, donor serum creatinine, recipient body mass index, re-transplantations beyond the second kidney transplantation, and cold ischemia time had an independent, significant influence on graded renal graft function 1 year after kidney transplantation. AUROC analysis of the prognostic model was >0.700 for all GFR categories except KDIGO G5, indicating high sensitivity and specificity of prediction. CONCLUSIONS For improvement of renal graft function, organs from older donors or donors with high serum creatinine should not be used in obese recipients and for re-transplantations beyond the second one. Cold ischemia time should be as short as possible.

Association between COVID-19 convalescent plasma antibody levels and COVID-19 outcomes stratified by clinical status at presentation

BackgroundThere is a need to understand the relationship between COVID-19 Convalescent Plasma (CCP) anti-SARS-CoV-2 IgG levels and clinical outcomes to optimize CCP use. This study aims to evaluate the relationship between recipient baseline clinical status, clinical outcomes, and CCP antibody levels.MethodsThe study analyzed data from the COMPILE study, a meta-analysis of pooled individual patient data from 8 randomized clinical trials (RCTs) assessing the efficacy of CCP vs. control, in adults hospitalized for COVID-19 who were not receiving mechanical ventilation at randomization. SARS-CoV-2 IgG levels, referred to as ‘dose’ of CCP treatment, were retrospectively measured in donor sera or the administered CCP, semi-quantitatively using the VITROS Anti-SARS-CoV-2 IgG chemiluminescent immunoassay (Ortho-Clinical Diagnostics) with a signal-to-cutoff ratio (S/Co). The association between CCP dose and outcomes was investigated, treating dose as either continuous or categorized (higher vs. lower vs. control), stratified by recipient oxygen supplementation status at presentation.ResultsA total of 1714 participants were included in the study, 1138 control- and 576 CCP-treated patients for whom donor CCP anti-SARS-CoV2 antibody levels were available from the COMPILE study. For participants not receiving oxygen supplementation at baseline, higher-dose CCP (/control) was associated with a reduced risk of ventilation or death at day 14 (OR = 0.19, 95% CrI: [0.02, 1.70], posterior probability Pr(OR < 1) = 0.93) and day 28 mortality (OR = 0.27 [0.02, 2.53], Pr(OR < 1) = 0.87), compared to lower-dose CCP (/control) (ventilation or death at day 14 OR = 0.79 [0.07, 6.87], Pr(OR < 1) = 0.58; and day 28 mortality OR = 1.11 [0.10, 10.49], Pr(OR < 1) = 0.46), exhibiting a consistently positive CCP dose effect on clinical outcomes. For participants receiving oxygen at baseline, the dose-outcome relationship was less clear, although a potential benefit for day 28 mortality was observed with higher-dose CCP (/control) (OR = 0.66 [0.36, 1.13], Pr(OR < 1) = 0.93) compared to lower-dose CCP (/control) (OR = 1.14 [0.73, 1.78], Pr(OR < 1) = 0.28).ConclusionHigher-dose CCP is associated with its effectiveness in patients not initially receiving oxygen supplementation, however, further research is needed to understand the interplay between CCP anti-SARS-CoV-2 IgG levels and clinical outcome in COVID-19 patients initially receiving oxygen supplementation.

The question of screening organ donors for hepatitis e virus: a case report of transmission by kidney transplantation in France and a review of the literature

BackgroundHepatitis E is a potentially serious infection in organ recipients, with an estimated two-thirds of cases becoming chronic, and with a subsequent risk of cirrhosis and death. In Europe, transmission occurs most often through the consumption of raw or undercooked pork, more rarely through blood transfusion, but also after solid organ transplantation. Here we describe a case of Hepatitis E virus (HEV) infection transmitted following kidney transplantation and review the literature describing cases of HEV infection transmitted by solid organ transplantation.Case presentationThree weeks after kidney transplantation, the patient presented with an isolated minimal increase in GGT and hepatic cytolysis 6 months later, leading to the diagnosis of genotype 3c hepatitis E, with a plasma viral load of 6.5 log10IU/mL. In retrospect, HEV RNA was detected in the patient's serum from the onset of hepatitis, and in the donor's serum on the day of donation, with 100% identity between the viral sequences, confirming donor-derived HEV infection. Hepatitis E had a chronic course, was treated by ribavirin, and relapsed 10 months after the end of treatment.DiscussionSeven cases of transmission of HEV by solid organ transplantation have been described since 2012 without systematic screening for donors, all diagnosed at the chronic infection stage; two patients died. HEV organ donor transmission may be underestimated and there is insufficient focus on immunocompromised patients in whom mild liver function test impairment is potentially related to hepatitis E. However, since HEV infection is potentially severe in these patients, and as evidence accumulates, we believe that systematic screening of organ donors should be implemented for deceased and living donors regardless of liver function abnormalities, as is already the case in the UK and Spain. In January 2024, the French regulatory agency of transplantation has implemented mandatory screening of organ donors for HEV RNA.

Impact of institutional variables on centre performance in long-term survival after heart transplant.

The gold standard metric for centre-level performance in orthotopic heart transplantation (OHT) is 1-year post-OHT survival. However, it is unclear whether centre performance at 1 year is predictive of longer-term outcomes. This study evaluated factors impacting longer-term centre-level performance in OHT. Patients who underwent OHT in the USA between 2010 and 2021 were identified using the United Network of Organ Sharing data registry. The primary outcome was 5-year survival conditional on 1-year survival following OHT. Multivariable Cox proportional hazard models assessed the impact of centre-level 1-year survival rates on 5-year survival rates. Mixed-effect models were used to evaluate between-centre variability in outcomes. Centre-level risk-adjusted 5-year mortality conditional on 1-year survival was not associated with centre-level 1-year survival rates [hazard ratio: 0.99 (0.97-1.01, P = 0.198)]. Predictors of 5-year mortality conditional on 1-year survival included black recipient race, pre-OHT serum creatinine, diabetes and donor age. In mixed-effect modelling, there was substantial variability between centres in 5-year mortality rates conditional on 1-year survival, a finding that persisted after controlling for recipient, donor and institutional factors (P < 0.001). In a crude analysis using Kaplan-Meier, the 5-year survival conditional on 1-year survival was: low volume: 86.5%, intermediate volume: 87.5%, high volume: 86.7% (log-rank P = 0.52). These measured variables only accounted for 21.4% of the between-centre variability in 5-year mortality conditional on 1-year survival. Centre-level risk-adjusted 1-year outcomes do not correlate with outcomes in the 1- to 5-year period following OHT. Further research is needed to determine what unmeasured centre-level factors contribute to longer-term outcomes in OHT.