Discrepancies between two D-dimer assays and impact on clinical decisions; a retrospective analysis of samples tested in community and hospital-based laboratories in Auckland.

AIM: In patients with suspected venous thromboembolism, an elevated D-dimer level provides an important branch-point in the management pathway. This study compared two D-dimer assays, INNOVANCE® DDimer (Innovance) and STA®-Liatest® D-Di Plus (Liatest), to assess potential impact on clinical management. METHOD: Reflecting current practice in Waitemata, Auckland, we compared paired samples from 805 patients referred to hospital following a community D-dimer test. Samples were determined to be positive or negative using a 500µg/L fibrinogen equivalent units (FEU), and age-adjusted cut-offs. RESULTS: In the Innovance assay, 2% of samples had a result <500µg/L FEU. In contrast, by Liatest, 18% were below 500µg/L. This positive bias of Innovance was amplified with use of age-adjusted cut-offs; 23% of samples with an elevated Innovance result showed a normal result by Liatest. On average, the Innovance values were 22% higher than Liatest. Results suggestive of interference from heterophile antibodies were seen in 6% of sample-pairs. CONCLUSION: Innovance D-dimer test yielded higher values than Liatest and experienced interference from suspected heterophile antibodies. Discrepancies in nearly a quarter of patients may be leading to substantial under or over investigation, inefficient use of resources and clinical confusion.

Simulated RBC antibody identification training panels created using SARS-CoV-2 kodecytes and immune plasma.

BACKGROUND: Training is essential to develop and maintain skills required to be a competent serologist, yet samples required to achieve this are often difficult to obtain. We evaluated the feasibility of SARS-CoV-2 peptide modified RBCs (1144-kodecytes) to develop simulated antibody screening and identification panels of reagent RBCs suitable for practical training, recognition, and grading of serologic reactions. STUDY DESIGN AND METHODS: RBCs from a single donor were modified into kodecytes using Kode Technology function-spacer-lipid constructs bearing a short SARS-CoV-2 peptide. Kodecytes and unmodified cells were then arranged in patterns representative of RBC antibody profiles as simulated antibody screening and identification reagent cell panels (SASID), and then tested against immune donor plasma samples containing SARS-CoV-2 antibodies. Manual tube and two different gel card serologic platforms were evaluated by routine techniques. SASID exemplars were created for antibodies including D, Cw , f (ce), Jka (strong, weak, dosing), mixtures of D + E, Jka + K, Fya + E, high and low frequency antibodies and a warm IgG autoantibody. RESULTS: Kodecytes (positive reactions) and unmodified cells (negative) when arranged and tested in appropriate patterns in SASID panels were able to mimic IgG antibody reactions, and were capable of measuring both accuracy and precision in reaction grading. CONCLUSIONS: Kodecytes can be used to rapidly create in-house simulated yet realistic antibody screening and identification panels suitable for large scale training in the recognition and grading of serologic reactions.

COVID-19 antibody screening with SARS-CoV-2 red cell kodecytes using routine serologic diagnostic platforms.

BACKGROUND: The Coronavirus disease 2019 (COVID-19) pandemic is having a major global impact, and the resultant response in the development of new diagnostics is unprecedented. The detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a role in managing the pandemic. We evaluated the feasibility of using SARS-CoV-2 peptide Kode Technology-modified red cells (C19-kodecytes) to develop an assay compatible with existing routine serologic platforms. STUDY DESIGN AND METHODS: A panel of eight unique red cells modified using Kode Technology function-spacer-lipid constructs and bearing short SARS-CoV-2 peptides was developed (C19-kodecyte assay). Kodecytes were tested against undiluted expected antibody-negative and -positive plasma samples in manual tube and three column agglutination technology (CAT) platforms. Parallel analysis with the same peptides in solid phase by enzyme immunoassays was performed. Evaluation samples included >120 expected negative blood donor samples and >140 COVID-19 convalescent plasma samples, with independent serologic analysis from two centers. RESULTS: Specificity (negative reaction rate against expected negative samples) in three different CAT platforms against novel C19-kodecytes was >91%, which correlated with published literature. Sensitivity (positive reaction rate against expected positive convalescent, PCR-confirmed samples) ranged from 82% to 97% compared to 77% with the Abbott Architect SARS-CoV-2 IgG assay. Manual tube serology was less sensitive than CAT. Enzyme immunoassay results with some Kode Technology constructs also had high sensitivity. CONCLUSIONS: C19-kodecytes are viable for use as serologic reagent red cells for the detection of SARS-CoV-2 antibody with routine blood antibody screening equipment.

A standardized kodecyte method to quantify ABO antibodies in undiluted plasma of patients before ABO-incompatible kidney transplantation.

BACKGROUND: The ABO transplantation barrier can be breached if antibody is reduced to low levels. Current serologic methods involve testing natural RBCs against dilutions of plasma to determine antibody levels, but these methods are poorly standardized and inherently error prone with consequent large inter- and intra laboratory variation. We evaluated the feasibility of using antigen-standardized kodecytes and undiluted plasma as an alternative method for antibody measurement in patients preparing for ABO-incompatible kidney transplantation. STUDY DESIGN AND METHODS: A panel of five kodecytes, bearing defined levels of synthetic blood group A type 2 antigen was developed (kodecyte assay) to show reaction patterns against undiluted plasma that were indicative of anti-A and anti-A,B levels. This panel was evaluated against the contemporary method of testing dilutions of plasma against A1 cells to determine titer (A1 cell assay) in both column agglutination and tube techniques. Evaluation samples included reference standards, 102 group O plus 23 group B donors, and 40 pre- and post-plasmapheresis samples from five prospective ABO-incompatible kidney transplant patients. RESULTS: Comparisons between the kodecyte and A1 cell assays found greater than 90% correlation for all samples. Tube and column agglutination technology platform differences were observed with A1 cells and kodecytes. Discordant samples were generally found to have high ratios of IgG:IgM or vice versa. CONCLUSIONS: The kodecyte assay is a simple method that requires no sample dilution, and an optimized two-cell kodecyte panel is potentially capable of informing ABO-incompatible kidney transplantation decisions based on antibody levels.

Blood Group O→A Transformation by Chemical Ligation of Erythrocytes.

Agglutination of red blood cells (RBCs) remains the only practical method for routine use for ABH typing in clinical practice. However, exact mechanistic details of agglutination are not yet thoroughly studied. In this research, RBCs of blood group O were converted to blood group A through two approaches: by chemical ligation of the cells' glycocalyx with synthetic blood group A tetrasaccharide, and by insertion of synthetic glycolipid carrying the same A antigen into the cells' membranes. The O→A ligated RBCs and natural A RBCs showed comparable agglutination characteristics with antibodies. As expected, RBCs with inserted glycolipid showed lower agglutination scores. This approach could help cell biologists in site-specific and cell-friendly modification of glycocalyx by other ligands.

Antibody complement-mediated hemolytic studies with kodecytes reveal that human complement utilized in the classical pathway is more stable than generally accepted.

BACKGROUND: Complement has significant status in the field of transfusion medicine. The accepted stability profile of complement is based on historical studies of diluted human serum hemolyzing rabbit heterophile antibody-sensitized sheep red blood cells (RBCs). Contemporary tools are available to reevaluate these historical observations using human heterophile antibodies, undiluted serum, and antigen-modified human RBCs. STUDY DESIGN AND METHODS: Human RBCs were made into "animal-like" kodecytes with heterophile Galα3Galß4GlcNAcß function-spacer-lipid constructs. These α-Gal-kodecytes were prepared with an antigen dilution capable of consistently producing 50% antibody-mediated hemolysis against human α1-3galactose heterophile antibodies and undiluted standardized serum. Standardized human serum aliquots from a two-donor pool stored at -85, -20, 4, 22, and 37°C for durations of up to 150 days were evaluated for loss of hemolytic activity. Where practical methodologic procedures were aligned with historical studies. RESULTS: Comparison of the historical assay with the α-Gal-kodecyte assay against complement activity standards showed concordance. However, in most scenarios complement was found to be more than twice as stable as generally accepted. At least 60% of complement hemolytic activity was observed in serum stored at 22°C for 1 week or 2 months at 4°C. No loss of hemolytic activity was observed after 5 months' storage at temperatures below -20°C. CONCLUSIONS: An alternative method using undiluted serum and modified human RBCs observed that classical-pathway complement hemolytic activity in stored human serum is at least twice as stable as previously accepted.

Training students in serologic reaction grading increased perceptions of self-efficacy and ability to recognize serologic reactions but decreased grading accuracy.

BACKGROUND: The ability to recognize and grade serologic reactions in manual techniques remains an important skill both for reference laboratories and in disaster-relocated laboratory services. Developing skills in recognizing and grading serologic reactions is limited to some extent by the range of samples available. STUDY DESIGN AND METHODS: Twenty-six students studying transfusion science were presented with blinded grading panels consisting of mixes of natural cells and kodecytes (natural cells modified with synthetic blood group antigens) representing a range of serologic grades. Results from 15-minute exercises over 17 contact weeks were assessed to determine if training with grading panels would have an impact on the ability of students to recognize and correctly grade serologic reactions. Twenty-one clinically active practitioners also took part in a single analysis. RESULTS: Grading exercises found that the use of kodecytes and natural negative cells were able to identify deficiencies in both students' and practitioners' ability to recognize negative and grade serologic reactions. The seventeen 15-minute exercises undertaken with students revealed that although there was some improvement in performance in recognizing positive and negative serologic reactions there was also a degradation in ability to accurately grade. Self-assessment showed a major improvement in students' self-efficacy. CONCLUSIONS: The use of serologic grading panels created with kodecytes was suitable as a tool to recognize and monitor serologic grading abilities. Evidence suggests that for both students and practitioners to gain and sustain competency in serologic reaction recognition and grading, they will require ongoing training and monitoring of competence.

Core temperature changes in resuspended red blood cells (RBCs) and pediatric RBCs removed from refrigerated storage.

BACKGROUND: The 30-minute rule, whereby intact red blood cell (RBC) products may be returned to stock if returned to 4 degrees C storage within 30 minutes of issue, was established many years ago. It was based on observations that the core temperature of units of whole blood removed from storage temperatures of 1 to 6 degrees C, and left at room temperature, would reach 10 degrees C at between 45 minutes and 1 hour. STUDY DESIGN AND METHODS: Forty-one units of RBCs resuspended leukoreduced and 8 units of pediatric RBCs resuspended leukoreduced were exposed to ambient temperature for periods of time between 0 and 60 minutes. Core temperatures of all units were measured at 1-minute or 5-minute intervals. RESULTS: Resuspended RBCs units reached a mean core temperature of 10 degrees C at 15 minutes, 12.7 degrees C at 30 minutes, and 15 degrees C at 60 minutes. Pediatric RBCs reached a mean core temperature of 12.8 degrees C at 15 minutes, 15.5 degrees C at 30 minutes, and 17.8 degrees C at 60 minutes. CONCLUSION: In view of our results, and the range of RBC products now available, it may be timely for blood services to review and reduce the 30-minute rule.

An intentional opiate intoxication of an infant: when medical toxicology and child maltreatment services merge.

We present an instructive case of a 5-week-old infant seen in the emergency department with acute inspiratory stridor and depressed level of consciousness. His emergency department course identified an acute opiate intoxication. The child also developed chest wall rigidity, a rare complication of narcotic use. We discuss the emergency department management, as well as the toxicologic and child protection investigations.