May 22, 2022

Performance of a flow cytometry-based immunoassay for detection of antibodies binding to SARS-CoV-2 spike protein

The performance of a laboratory-developed IgG/IgA flow cytometry-based immunoassay (FCI) using Jurkat T cells stably expressing full-length native S protein was compared against Elecsys electrochemiluminiscent (ECLIA) Anti-SARS-CoV-2 S (Roche Diagnostics, Pleasanton, CA, USA), and Liaison SARS-CoV-2 TrimericS IgG chemiluminiscent assay (CLIA) (Diasorin S.p.a, Saluggia, IT) for detection of SARS-CoV-2-specific antibodies. A total of 225 serum/plasma specimens from 120 acute or convalescent COVID-19 individuals were included. Overall, IgG/IgA-FCI yielded the highest number of positives (n = 179), followed by IgA-FCI (n = 177), Roche ECLIA (n = 175), IgG-FCI (n = 172) and Diasorin CLIA (n = 154).
For sera collected early after the onset of symptoms (within 15 days) IgG/IgA-FCI also returned the highest number of positive results (52/72; 72.2%). Positive percent agreement between FCI and compared immunoassays was highest for Roche ECLIA, ranging from 96.1 (IgG/IgA-FCI) to 97.7% (IgG-FCI), whereas negative percent agreement was higher between FCI and Diasosin CLIA, regardless of antibody isotype. The data suggest that FCI may outperform Roche ECLIA and Diasorin CLIA in terms of clinical sensitivity for serological diagnosis of SARS-CoV-2 infection.

Potential for Process Improvement of Clinical Flow Cytometry by Incorporating Real-Time Automated Screening of Data to Expedite Addition of Antibody Panels

Objectives: We desired an automated approach to expedite ordering additional antibody panels in our clinical flow cytometry lab. This addition could improve turnaround times, decrease time spent revisiting cases, and improve consistency.
Methods: We trained a machine learning classifier to use our screening B-cell panel to predict whether we should order an additional panel to distinguish chronic lymphocytic lymphoma from mantle cell lymphoma. We used data from 2016 to 2018 for training and validation, and cases were restricted to the first case per patient (9,635 cases, 887 with the additional panel). We applied the model in real time over approximately 2.5 months in 2020 to 376 sequential cases, with automated email notifications for positive predictions.
Results: Using 80% of the data from 2016 to 2018 to train and 20% for validation, we achieved 95% area under the receiving operating characteristic curve (AUROC) and 94% accuracy in the validation set. Applying the classifier in real time achieved 89% AUROC and 94% real-time prediction accuracy (precision [positive predictive value] = 51%, recall [sensitivity] = 78%, and F1 score = 0.62). Fourteen of the 17 false positives had prior diagnoses to which the algorithm was not privy.
Conclusions: As an observational, not interventional study, our system performed well on testing within our laboratory for identifying cases to be flagged but cannot be used without laboratory-specific modifications.

Validation of a Flow Cytometry Live Cell-Based Assay to Detect Myelin Oligodendrocyte Glycoprotein Antibodies for Clinical Diagnostics

Background: Myelin oligodendrocyte glycoprotein antibodies (MOG Ab) are essential in the diagnosis of MOG Ab-associated disease (MOGAD). Live cell-based assays (CBAs) are the gold standard for MOG Ab detection with improved sensitivity and specificity over fixed CBAs. A number of testing centers have used flow cytometry for its high throughput and quantitative utility. Presently, there is increasing demand to translate these research-based methods into an accredited routine diagnostic setting.
Methods: A flow cytometry live CBA was used to detect MOG Ab in patients with demyelination. Serostatuses were compared between a research-based assay and a streamlined diagnostic assay. Inter-laboratory validation of the streamlined assay was performed in an accredited diagnostic laboratory. Further streamlining was performed by introducing a borderline serostatus range and reducing the number of controls used to determine the positivity threshold.
Results: High serostatus agreement (98%-100%) was observed between streamlined and research-based assays. Intra- and inter-assay imprecision was improved in the streamlined assay (mean intra- and inter-assay CV = 7.3% and 27.8%, respectively) compared to the research-based assay (mean intra- and inter-assay CV = 11.8% and 33.6%, respectively). Borderline positive and clear positive serostatuses were associated with confirmed phenotypes typical of MOGAD. Compared to using 24 controls, robust serostatus classification was observed when using 13 controls without compromising analytical performance (93%-98.5% agreement).
Conclusions: Flow cytometry live CBAs show robust utility in determining MOG Ab serostatus. Streamlining and standardizing use of this assay for diagnostics would improve the accuracy and reliability of routine testing to aid diagnosis and treatment of patients with demyelination.

Passive antibody transfer on human leukocytes: application to small animal allergy diagnosis by flow cytometry

Animal allergy diagnosis is based mainly on clinical history, skin tests and, at least for dogs, specific IgE antibodies. The quality of anti-canine IgE antibodies is variable and monoclonal antibodies have been recently characterized. The allergen panel tested in humans and in dogs is similar except for flea and for Staphylococcus. Allergen-induced basophil activation may be measured by the release of mediators such as histamine and leukotriene C4 and by the expression of the CD63 marker on basophil membrane. This latter method is based on the flow cytometric analysis of leukocyte suspensions after double anti-IgE FITC, anti-CD63 PE labelling of human basophils, and has been validated for aero-allergens, food allergens, venoms and several drugs for human allergy diagnosis.
After having demonstrated that, in the dog, anaphylactic anti- bodies were capable of binding to human basophil high-affinity receptors for IgE, we went up a flow cytometric method for animal allergy diagnosis based on passive sensitization of human basophils. Prelim- inary results obtained by this method for allergens such as house dust mite or pollen were very encouraging. This method is faster and less expensive than the methods based on mediator release but is still dependent on the availability of fresh human leukocytes. This method may represent a new sensitive and specific method for animal allergy diagnosis.

Human ADAMTS13 AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS16951M AssayPro Kit 693 EUR

Human PEBP1 AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS32551M AssayPro Kit 693 EUR

Human SOD2 AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS32611M AssayPro Kit 693 EUR

Human ARA9 AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS32855M AssayPro Kit 693 EUR

Human ADPRH AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS32900M AssayPro Kit 693 EUR

Human AHA1 AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS32922M AssayPro Kit 693 EUR

Human ACAD8 AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS33059M AssayPro Kit 693 EUR

Human ADK AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS33145M AssayPro Kit 693 EUR

Human Cystathionase AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS35613M AssayPro Kit 693 EUR

Human Myoglobin (Mb) AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS11552M AssayPro Kit 693 EUR

Human Enolase 1 AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS32014M AssayPro Kit 693 EUR

Human Aldolase C AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS32088M AssayPro Kit 693 EUR

Human HB-EGF AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS33171M AssayPro Kit 693 EUR

Flow Cytometry Fixation/Permeabilization Kit

23006 Biotium 50test 80 EUR

Human Thioredoxin-1 (TRX1) AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS13016M AssayPro Kit 693 EUR

Human Immunoglobulin G3 (IgG3) AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS24107M AssayPro Kit 693 EUR

Human Immunoglobulin G4 (IgG4) AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS24108M AssayPro Kit 693 EUR

Human Annexin A4 (ANXA4) AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS30112M AssayPro Kit 693 EUR

Human Alcohol Dehydrogenase (AKR1A1) AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS32007M AssayPro Kit 693 EUR

Human Annexin A1 (ANXA1) AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS32171M AssayPro Kit 693 EUR

Human Diazepam Binding Inhibitor AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS32991M AssayPro Kit 693 EUR

Human Annexin A3 (ANXA3) AssayLite Multi-Color Conjugated Antibodies Flow Cytometry Kit

FACS33118M AssayPro Kit 693 EUR

Evaluation of commercially available antibodies and fluorescent conotoxins for the detection of surface ganglionic acetylcholine receptor on the neuroblastoma cell line, IMR-32 by flow cytometry

Commercially available antibodies that bind to the human muscle acetylcholine receptor (ACHR) have been validated previously for flow cytometric use (Keefe et al., 2009; Leite et al., 2008; Lozier et al., 2015). Despite a multitude of commercially available antibodies to other nicotinic ACHRs, validation in a wide variety of immunoassay formats is lacking; when studied, a large proportion of these antibodies have been deemed not fit for most research purposes (Garg and Loring, 2017). We have recently described a flow cytometric immunomodulation assay for the diagnosis of Autoimmune Autonomic Ganglionopathy (AAG) (Urriola et al., 2021) that utilises the monoclonal antibody mab35(Urriola et al., 2021) which is specific for ganglionic ACHR (gnACHR) that contain α3 subunits (Vernino et al., 1998a).
Other fluorescent ligands for α3-gnACHR have not been validated for flow cytometric use. We investigated 7 commercially sourced antibodies and 3 synthetic fluorescent novel conotoxins purported to specifically bind to the extracellular domains of the gnACHR, and compared the results to staining by mab35, using flow cytometry with the neuroblastoma cell line IMR-32. We also evaluated the degree of non-specific binding by depleting the cell membrane of the relevant acetylcholine receptor with a pre-incubation step involving the serum from a patient with Autoimmune Autonomic Ganglionopathy containing pathogenic antibodies to the ganglionic acetylcholine receptor. None of the assessed conotoxins, and only one antibody (mab35) was found to perform adequately in flow cytometric staining of the native ganglionic acetylcholine receptor.

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