Cross-validation of ELISA and a portable surface plasmon resonance instrument for IgG antibody serology with SARS-CoV-2 positive individuals.

We report on the development of surface plasmon resonance (SPR) sensors and matching ELISAs for the detection of nucleocapsid and spike antibodies specific against the novel coronavirus 2019 (SARS-CoV-2) in human serum, plasma and dried blood spots (DBS). When exposed to SARS-CoV-2 or a vaccine against SARS-CoV-2, the immune system responds by expressing antibodies at levels that can be detected and monitored to identify the fraction of the population potentially immunized against SARS-CoV-2 and support efforts to deploy a vaccine strategically. A SPR sensor coated with a peptide monolayer and functionalized with various sources of SARS-CoV-2 recombinant proteins expressed in different cell lines detected human anti-SARS-CoV-2 IgG antibodies in clinical samples. Nucleocapsid expressed in different cell lines did not significantly change the sensitivity of the assays, whereas the use of a CHO cell line to express spike ectodomain led to excellent performance. This bioassay was performed on a portable SPR instrument capable of measuring 4 biological samples within 30 minutes of sample/sensor contact and the chip could be regenerated at least 9 times. Multi-site validation was then performed with in-house and commercial ELISA, which revealed excellent cross-correlations with Pearson's coefficients exceeding 0.85 in all cases, for measurements in DBS and plasma. This strategy paves the way to point-of-care and rapid testing for antibodies in the context of viral infection and vaccine efficacy monitoring.

Comparison of the Effect of Holder Pasteurization and High-Pressure Processing on Human Milk Bacterial Load and Bioactive Factors Preservation.

OBJECTIVES: This project aims at comparing the impact of Holder pasteurization (HoP) and high-pressure processing (HPP) on bacterial load and retention of immunological components in human milk. METHODS: Human milk samples discarded by the Public Mothers' milk bank (Montreal, Canada) for bacterial purpose were pooled (n = 6) and pasteurized either by heating in a water bath (62.5°C, 30 minutes) or by HPP treatment (425 MPa, four cycles of 6 minutes, initial milk temperature of 4°C or 37°C). Bacterial load, lysozyme activity, and levels of immunoglobulins, lactoferrin, lipase, and 26 cytokines were analyzed. Untreated milk samples from same pools served as control. RESULTS: HPP treatment of milk allows a similar elimination of bacteria than HoP; bacterial counts were under the detection limit [<3 colony-forming units (CFU)/mL] in 50% of milk pools after HPP treatment, compared to 17% for HoP. With initial heating of samples to 37°C before HPP treatment, inactivation to an extent under the detection limit was reached in 67% of pools. There is no significant difference in IgA, lysozyme, and cytokines concentrations between untreated milk and all treatment methods. While no significant difference was observed in the amount of lipase (P > 0.07) and IgG (P > 0.11) between untreated milk and HPP-treated milk samples, HoP seems to be damaging for these factors (P < 0.04). IgM is well preserved in HPP-4°C samples compared to untreated milk (P = 0.07) whereas a decrease is observed for this immunoglobulin levels in HPP-37°C and HoP samples (P < 0.01). Lactoferrin activity, is well maintained in HPP-37°C milk samples in comparison to untreated milk samples (P = 0.52). A decrease in activity of this molecule is noted for samples treated with HPP at 4°C (P = 0.02) and this decrease is even more pronounced for HoP samples (P = 0.004). CONCLUSIONS: HPP is a promising alternative to HoP for treatment of human milk intended to preterm babies. Our results demonstrate that HPP treatment of human milk provides safe milk with less detrimental effects on the biochemically and immunologically active milk components than HoP.

Rhesus D Antigenic Determinants on Residual Red Blood Cells in Apheresis and Buffy Coat Platelet Concentrates.

BACKGROUND: The level of residual red blood cells (RBCs) in platelet concentrates (PCs) is of interest because of clinical concerns related to alloimmunization to RBC antigens in transfused patients. This work aims at characterizing and quantifying the levels of intact and fragmented RBCs in apheresis (AP-PCs) and buffy coat PCs (BC-PCs) to assess their potential risk for RhD antigen alloimmunization. METHODS: After staining with anti-CD41 (platelets) and anti-CD235a (RBCs) antibodies, the size and density of RhD antigen on intact and fragmented RBCs were analyzed by flow cytometry. RESULTS: Residual RBC counts were 29 ± 22 × 106/unit in AP-PCs and 121 ± 54 × 106/unit in BC-PCs, which correspond to about 3 and 11 µL of RBCs by product, respectively. RhD expression was about 4 times higher on RBC particles in AP-PCs, and these particles contribute to 66 and 75% of the total antigenic load in BC-PCs and AP-PCs, respectively. CONCLUSIONS: Processing methods influence the quantity and nature of contaminating residual RBCs and RBC-derived particles in PCs. The estimation of residual RBCs in these blood products is generally based on measurements of intact RBCs, which might underestimate the risk for alloim-munization in transfused patients. The question of whether these RBC-derived particles can produce an immune response and, thus, should then be taken into consideration for Rh immune prophylactic treatments, remains to be clarified.

The impact of red blood cell manufacturing variables on bacterial growth dynamics: a pilot study.

BACKGROUND AND OBJECTIVES: Bacterial contamination of red blood cells (RBC) remains a rare but serious clinical concern. Despite the low temperature storage of RBC, some bacteria can proliferate. The impact of RBC additive solutions (AS), manufacturing method or donor sex on bacterial growth/survival in RBC was addressed in this pilot study. MATERIALS AND METHODS: Using a partial pool-and-split design, bacterial growth/survival was assessed in intentionally inoculated RBC, manufactured separately from male and female donors using three different manufacturing methods (two whole blood [WB] filtration methods; one RBC filtration method), and resuspended in one of four AS: SAGM, PAGGSM, AS-1 or AS-3. At the beginning of storage, RBC were inoculated with 10 CFU/ml of either Klebsiella pneumoniae, Staphylococcus epidermidis, Yersinia enterocolitica or Propionibacterium acnes. Manufacturing, inoculation, storage (until day 42) and monitoring of bacterial growth were conducted at two sites: Canadian Blood Services and Héma-Québec. RESULTS: Yersinia enterocolitica was the only bacterium that proliferated during storage at both sites. RBC tested at Canadian Blood Services had higher bacterial concentrations than those at Héma-Québec (P = 0·0044). At Héma-Québec, where two different manufacturing methods were used, Y. enterocolitica reached significantly higher bacterial concentrations in AS-3 RBC (WB filtration method) compared to units prepared in the other three AS (RBC filtration method; P < 0·05). Bacterial survival/growth dependent on donor sex was not uniformly noted. CONCLUSION: Only one of four bacteria grew under RBC storage conditions. The results indicate that RBC manufacturing variables, rather than AS or donor sex, affect bacterial growth in RBC.

Bordetella holmesii Contamination of Platelet Concentrates: Revisiting the Definition of a Positive Culture.

Bacterial contamination remains the most important infectious risk of platelet transfusion. After an initially positive result, a second test is performed on the blood products and the initial culture bottle to confirm the contamination. Based on the blood center's decision algorithm used, results can be either confirmed negative, positive, or indeterminate, or be unconfirmed or discordant. Here, we report the first cases of platelet concentrates contaminated with Bordetella holmesii The in vitro growth characteristics of this unusual contaminant in platelet concentrate were investigated. Two B. holmesii strains isolated from platelet concentrates, as well as a control strain (Serratia marcescens), were spiked into platelet concentrates (PCs) at 1 and 10 CFU/ml. PCs were stored at 20 to 24°C under agitation. Samples were collected on days 2, 3, 4, and 7 for colony count and for bacterial screening using the BacT/Alert 3D system. Two PCs were detected as being positive for B. holmesii However, recultures were negative. In vitro, B. holmesii did not grow but remained detectable in PCs. Its viability diminished rapidly in contact with human plasma. Upon screening using the BacT/Alert 3D system, the majority of products spiked with B. holmesii were negative. This is the first description of PCs contaminated with B. holmesii This bacterium survives in blood products and remains dormant at low concentrations in blood products stored at room temperature, thus making difficult its detection with the BacT/Alert 3D system. The present definition of a true-positive culture of PCs may be overly restrictive for certain bacterial strains.

Optimization of cord blood unit sterility testing: impact of dilution, analysis delay, and inhibitory substances.

BACKGROUND: Different methods are used by cord blood banks to prepare samples for sterility testing. Suboptimal methods can result in the release of contaminated products. In our organization, samples are prepared by diluting the final product in RPMI-1640 medium. In this work, we have compared our method with different approaches to verify whether optimization should be sought. STUDY DESIGN AND METHODS: Cord blood units (n = 6 units per bacterial strain) characterized to contain inhibitory substances or not were inoculated (10 colony-forming units/mL) with Streptococcus agalactiae, Staphylococcus epidermidis, Klebsiella pneumoniae, Escherichia coli, or Bacteroides fragilis. After plasma and red blood cell removal, stem cell concentrates were diluted in RPMI-1640, thioglycollate, or the unit's plasma. These products, as well as final product, plasma, and red blood cell fractions, were held from 0 to 72 hours at 20 to 24°C before inoculation in culture bottles and detection using the BacT/ALERT 3D system. RESULTS: Dilution of cell concentrates in RPMI-1640 allowed bacterial detection in 93.3% of noninhibitory cord blood samples after a 24-hour storage period. Thioglycollate medium better promoted bacterial growth in inhibitory cord blood samples that were held for 72 hours before testing (66.7%) compared with RPMI-1640 (45.0%). Less than 33% of all spiked plasma samples were detected by the BacT/ALERT 3D system. CONCLUSION: Diluting cord blood samples in culture medium containing bacterial growth promoting substances is a suitable option for sterility testing, whereas the use of plasma should be proscribed, because it might lead to false-negative results. Because inhibitory substances affect bacterial growth, inoculation of culture bottles should be done rapidly after sample preparation.

Assessment of the longitudinal humoral response in non-hospitalized SARS-CoV-2-positive individuals at decentralized sites: Outcomes and concordance.

Introduction: Early in the COVID-19 pandemic, reagent availability was not uniform, and infrastructure had to be urgently adapted to undertake COVID-19 surveillance. Methods: Before the validation of centralized testing, two enzyme-linked immunosorbent assays (ELISA) were established independently at two decentralized sites using different reagents and instrumentation. We compared the results of these assays to assess the longitudinal humoral response of SARS-CoV-2-positive (i.e., PCR-confirmed), non-hospitalized individuals with mild to moderate symptoms, who had contracted SARSCoV-2 prior to the appearance of variants of concern in Québec, Canada. Results: The two assays exhibited a high degree of concordance to identify seropositive individuals, thus validating the robustness of the methods. The results also confirmed that serum immunoglobulins persist ≥ 6 months post-infection among non-hospitalized adults and that the antibodies elicited by infection cross-reacted with the antigens from P.1 (Gamma) and B.1.617.2 (Delta) variants of concern. Discussion: Together, these results demonstrate that immune surveillance assays can be rapidly and reliably established when centralized testing is not available or not yet validated, allowing for robust immune surveillance.

Cross-reactivity of antibodies from non-hospitalized COVID-19 positive individuals against the native, B.1.351, B.1.617.2, and P.1 SARS-CoV-2 spike proteins.

SARS-CoV-2 variants of concern (VOCs) have emerged worldwide, with implications on the spread of the pandemic. Characterizing the cross-reactivity of antibodies against these VOCs is necessary to understand the humoral response of non-hospitalized individuals previously infected with SARS-CoV-2, a population that remains understudied. Thirty-two SARS-CoV-2-positive (PCR-confirmed) and non-hospitalized Canadian adults were enrolled 14-21 days post-diagnosis in 2020, before the emergence of the B.1.351 (also known as Beta), B.1.617.2 (Delta) and P.1 (Gamma) VOCs. Sera were collected 4 and 16 weeks post-diagnosis. Antibody levels and pseudo-neutralization of the ectodomain of SARS-CoV-2 spike protein/human ACE-2 receptor interaction were analyzed with native, B.1.351, B.1.617.2 and P.1 variant spike proteins. Despite a lower response observed for the variant spike proteins, we report evidence of a sustained humoral response against native, B.1.351, B.1.617.2 and P.1 variant spike proteins among non-hospitalized Canadian adults. Furthermore, this response inhibited the interaction between the spike proteins from the different VOCs and ACE-2 receptor for ≥ 16 weeks post-diagnosis, except for individuals aged 18-49 years who showed no inhibition of the interaction between B.1.617.1 or B.1.617.2 spike and ACE-2. Interestingly, the affinity (KD) measured between the spike proteins (native, B.1.351, B.1.617.2 and P.1) and antibodies elicited in sera of infected and vaccinated (BNT162b2 and ChAdOx1 nCoV-19) individuals was invariant. Relative to sera from vaccine-naïve (and previously infected) individuals, sera from vaccinated individuals had higher antibody levels (as measured with label-free SPR) and more efficiently inhibited the spike-ACE-2 interactions, even among individuals aged 18-49 years, showing the effectiveness of vaccination.

Evaluation of the Tempo® System: Improving the Microbiological Quality Monitoring of Human Milk.

Background: Bacteriological testing of donor human milk is mostly done both before and after pasteurization to control contamination in the end-product and meet the microbiological standards. Although the plate count method represents a reliable and sensitive technique and is considered the gold standard for bacteriological testing, it is recognized for being time-consuming and requiring qualified personnel. Recently, faster testing technologies, mostly geared toward the food industry, have been developed. Among these, the bioMérieux TEMPO® system uses the most probable number method to assess microbiological content in a semi-automated fashion. Objective: The performances of the TEMPO® system in enumerating bacterial quality indicators in human milk were assessed and compared to the reference plate count method. Methods: Naturally and artificially contaminated human milk samples were used to compare the analytical performances of the TEMPO® system to the plate count technique. More specifically, bacteria belonging to the genera Bacillus, Enterobacteriaceae, Staphylococcus aureus, and total aerobic flora were screened using both methods. Bacteria isolated on agar plates containing selective media were identified by supplemental testing. Bacterial testing results and method parameters were compared using linear regression analyses and Bland-Altman approaches. Results: Naturally contaminated milk samples (n = 55) tested for total aerobic flora showed < 1 log (CFU/ml) discrepancy between the two methods in the output results for 98% of the samples. Comparative linear regression analyses demonstrate good correlations between the two methods (R 2 > 0.9). At lower levels of bacterial contamination, the TEMPO® method precision (C.V. < 8%) and accuracy (> 83%) were comparable to plate counts. Conclusions: The analytical performances of the TEMPO® system for human milk bacteriological testing are equivalent to the reference plate count method. Results from the TEMPO® system are available within a 24-h turnaround time from sample inoculation without the need for further supplemental testing, suggesting that this semi-automated method could be implemented within milk bank operations as an in-process monitoring technology to optimize end-product quality and safety.

Establishment of an immunoglobulin A-deficient blood donor registry with a simple in-house screening enzyme-linked immunosorbent assay.

BACKGROUND: Transfusion of blood products to immunoglobulin A (IgA)-deficient patients who have developed IgA antibodies can result in serious adverse reactions. To prepare compatible blood components for these patients, blood centers usually maintain a list of IgA-deficient blood donors. An in-house enzyme-linked immunosorbent assay (ELISA) was used to identify new IgA-deficient blood donors. STUDY DESIGN AND METHODS: An in-house ELISA was used to screen blood samples. IgA-deficient samples, defined as an IgA level below 0.05 mg per dL, were sent to the American Red Cross for confirmatory testing. RESULTS: Seventy-three confirmed IgA-deficient blood donors were identified among 38,759 screened blood donor samples (frequency, 1:531). IgA antibodies were found in 39 of these 73 blood donors (53%), although only 9 donors had a history of adult IgA exposure (transfusion or pregnancy). CONCLUSIONS: With a simple in-house ELISA, 73 blood donors were identified as IgA-deficient. From this number, 34 donors, without detectable anti-IgA in their plasma, were added to our IgA-deficient blood donor panel to maximize the management of our inventory of IgA-deficient frozen blood components.

Characterization of blood components prepared from whole-blood donations after a 24-hour hold with the platelet-rich plasma method.

BACKGROUND: The preparation of platelet (PLT) concentrates (PCs) from PLT-rich plasma (PRP) requires that whole blood (WB) be processed within 8 hours of collection. Increasing WB storage time to 24 hours would be logistically attractive. This study compares the in vitro quality of blood components prepared from WB stored for 8 and 24 hours at room temperature before processing with the PRP method. STUDY DESIGN AND METHODS: WB units were collected from ABO-matched blood donors. To reduce individual variations, paired donations were drawn in parallel, pooled, and split back in the collection bag. One unit was held for 6 to 8 hours and the other for 22 to 24 hours at 20 to 24 degrees C. Prestorage leukoreduced components were prepared with the PRP as intermediate product and analyzed during storage. RESULTS: RBC units prepared after an 8- or 24-hour hold were comparable in terms of hemolysis, sodium, pH, and ATP levels. RBC 2,3- diphosphoglycerate (2,3-DPG) was significantly lower in RBCs prepared from 24-hour hold donations immediately after processing but not after 20 days of storage. Residual white blood cells were approximately fivefold higher (p < 0.05) in 24-hour RBC units. For PCs, measurements for glucose, ATP, lactate, pH, extent of shape change, hypotonic shock response, and CD62p activation were similar. No differences were observed in the von Willebrand factor, factor (F)V, FVIII, and fibrinogen content of fresh-frozen plasma. CONCLUSIONS: The decrease in FVIII and RBC 2,3-DPG can be acceptable as a compromise to improve blood component logistics, but leukoreduction efficiency must be improved before considering the adoption of an overnight storage of WB before PRP processing.

Intravenous immunoglobulins induce the in vitro differentiation of human B lymphocytes and the secretion of IgG.

The therapeutic effects of intravenous immunoglobulins (IVIGs) in several autoimmune diseases are characterized by a decrease in pathologic autoantibody levels. Although little direct evidence has been reported in humans, the large repertoire of natural immunoglobulin G (IgG) antibodies in IVIGs is expected to be involved in the regulation of autoreactive B lymphocytes. In normal adult mice, IVIGs have been reported to modulate immature B cells as well as peripheral B lymphocytes through V-region connections. Studies with human serum also indicated that anti-idiotypic antibodies, present in IVIG preparations, could recognize both natural and pathologic autoantibodies. We have used an in vitro culture system to characterize the direct effect of IVIGs on human B lymphocytes. This in vitro culture system involves CD40 activation of B lymphocytes by its ligand CD154 in the presence of cytokines. In this system, addition of IVIGs decreased by 50% to 80% the expansion of B lymphocytes. This reduced expansion was due to a decrease in the proliferation rate. In addition, a portion of B lymphocytes was differentiated into IgG-secreting cells in the presence of IVIGs and the secreted IgGs were reactive with antigens such as nucleoprotamine, dsDNA, tetanus toxin, and human IgG F(ab')(2) fragments. These observations indicate that IVIGs can have direct effects on B lymphocytes and suggest that such IVIG regulation of B lymphocytes could be involved in the therapeutic effects of IVIGs in autoimmune diseases.