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This retrospective cross-sectional study investigates the impact of laboratory-specific hemoglobin reference intervals on electronic consultation (eConsult) referral patterns for suspected anemia and elevated hemoglobin at a tertiary care center in London, Ontario that serves Southwestern Ontario. The study analyzed referrals through the Ontario Telemedicine Network's eConsult platform for hemoglobin abnormalities, excluding patients under 18 years old, between July 1, 2019, and June 30, 2023.The main outcome measures were influence of hemoglobin reference intervals on the referral patterns for suspected anemia and elevated hemoglobin, as well as the extent of pre-referral laboratory testing. Of the 619 eConsults reviewed, 251 referrals for suspected anemia and 93 for elevated hemoglobin were analyzed. Referral patterns showed significant variance in hemoglobin levels based on different laboratory thresholds. Referrals for suspected anemia in females from laboratories whose lower limit was 120 g/L or greater had a hemoglobin concentration 7.5 g/L greater than referrals that used laboratories with a threshold lower than 120 g/L. The study also identified potential areas for improvement in pre-referral investigations; 44% of eConsults did not provide a ferritin level, 53% were missing a B12 level, and 81% were missing a reticulocyte count. In conclusion, laboratory reference intervals for hemoglobin significantly influence referral patterns for suspected hemoglobin abnormalities in Ontario's eConsult system. There is a need for standardized reference intervals and comprehensive pre-referral testing to avoid unnecessary medicalization and referrals. We propose an anemia management algorithm to guide primary care providers in the pre-referral investigation process.
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Myelodysplastic neoplasms (MDS) with ring sideroblasts (RS) are diagnosed via bone marrow aspiration in the presence of either (i) ≥15% RS or (ii) 5-14% RS and an SF3B1 mutation. In the MEDALIST trial and in an interim analysis of the COMMANDS trial, lower-risk MDS-RS patients had decreased transfusion dependency with luspatercept treatment. A total of 6817 patients with suspected hematologic malignancies underwent molecular testing using a next-generation-sequencing-based genetic assay and 395 MDS patients, seen at our centre from 1 January 2018 to 31 May 2023, were reviewed. Of these, we identified 39 evaluable patients as having lower-risk MDS with SF3B1 mutations: there were 20 (51.3%) males and 19 (48.7%) females, with a median age of 77 years (range of 57 to 92). Nineteen (48.7%) patients had an isolated SF3B1 mutation with a mean variant allele frequency of 35.2% +/- 8.1%, ranging from 7.4% to 46.0%. There were 29 (74.4%) patients with ≥15% RS, 6 (15.4%) with 5 to 14% RS, one (2.6%) with 1% RS, and 3 (7.7%) with no RS. Our study suggests that a quarter of patients would be missed based on the morphologic criterion of only using RS greater than 15% and supports the revised 2022 definitions of the World Health Organization (WHO) and International Consensus Classification (ICC), which shift toward molecularly defined subtypes of MDS and appropriate testing.
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Mutação , Síndromes Mielodisplásicas , Fosfoproteínas , Fatores de Processamento de RNA , Organização Mundial da Saúde , Humanos , Fatores de Processamento de RNA/genética , Masculino , Feminino , Idoso , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/classificação , Pessoa de Meia-Idade , Estudos Retrospectivos , Idoso de 80 Anos ou mais , Fosfoproteínas/genética , Anemia Sideroblástica/genéticaRESUMO
Background: Thrombocytosis is a common reason for referral to Hematology. Differentiating between secondary causes of thrombocytosis and essential thrombocythemia (ET) is often clinically challenging. A practical diagnostic approach to identify secondary thrombocytosis could reduce overinvestigation such as next generation sequencing (NGS) panel. Methods and Results: All adult patients with thrombocytosis (≥450 × 109/L) who underwent molecular testing at a single tertiary care centre between January 1, 2018 and May 31, 2021 were evaluated. Clinical and laboratory variables were compared between patients with secondary thrombocytosis vs. ET. Clinical variables included smoking, thrombosis, splenectomy, active malignancy, chronic inflammatory disease, and iron deficiency anemia. Laboratory variables included complete blood count (CBC), ferritin, and myeloid mutations detected by NGS. The overall yield of molecular testing was 52.4%; 92.1% of which were mutations in JAK2, CALR, and/or MPL. Clinical factors predictive of ET included history of arterial thrombosis (p < 0.05); active malignancy, chronic inflammatory disease, splenectomy, and iron deficiency were associated with secondary thrombocytosis (p < 0.05). A diagnosis of ET was associated with higher hemoglobin, mean corpuscular volume (MCV), red cell distribution width (RDW), and mean platelet volume (MPV), while secondary thrombocytosis was associated with higher body mass index, white blood cells, and neutrophils (p < 0.01). Conclusion: A practical approach to investigating patients with persistent thrombocytosis based on clinical characteristics such as active malignancy, chronic inflammatory disease, splenectomy, and iron deficiency may assist in accurately identifying patients more likely to have secondary causes of thrombocytosis and reduce overinvestigation, particularly costly molecular testing.
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BACKGROUND: Patients with hematological cancers have increased COVID-19 morbidity and mortality, and these patients show attenuated vaccine responses. This study aimed to characterize the longitudinal humoral immune responses to COVID-19 vaccination in patients with hematological malignancies. PATIENTS AND METHODS: We conducted a prospective cohort study, collecting samples from March 2021 to July 2022, from patients seen at a cancer treatment center in London, Ontario, Canada, who met the following eligibility criteria: age ≥18 years, diagnosed with a hematological malignancy, recipient of a COVID-19 vaccine during the study period, and able to provide informed consent. RESULTS: Median anti-S titers (MST) were 0.0, 64.0, and 680.5 U/mL following first (V1), second (V2), and third (V3) vaccine doses, respectively. Patients with lymphoid malignancies' response to vaccination was attenuated compared to myeloid malignancy patients after V2 and V3 (P < .001, P < .01). Active treatment was associated with lower antibody titers (MST 10) compared to treatment 12-24 months (MST 465, P = .04367) and >24 months (MST 1660.5, P = .0025) prior to vaccination. V3 significantly increased antibody titers compared to V2 for patients less than 3 months from treatment. Increasing age was associated with smaller antibody response following V2 (P < .05), but not following V3. Patients receiving anti-CD20 therapy did not demonstrate increased antibody titer levels after V3 (V2 MST 0, V3 MST 0; P > .05). CONCLUSION: We report an attenuated serologic response to COVID-19 vaccination in our study population of patients with hematological malignancy. The immune response to vaccination was affected by patient age, diagnosis, treatment, and timing of treatment exposure.
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Vacinas contra COVID-19 , COVID-19 , Neoplasias Hematológicas , SARS-CoV-2 , Humanos , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/complicações , Masculino , Feminino , Estudos Prospectivos , Pessoa de Meia-Idade , COVID-19/prevenção & controle , COVID-19/imunologia , COVID-19/complicações , Idoso , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/uso terapêutico , SARS-CoV-2/imunologia , Adulto , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Vacinação , Idoso de 80 Anos ou mais , Imunidade HumoralAssuntos
Diabetes Mellitus Tipo 2 , Policitemia , Inibidores do Transportador 2 de Sódio-Glicose , Humanos , Inibidores do Transportador 2 de Sódio-Glicose/efeitos adversos , Estudos Retrospectivos , Policitemia/induzido quimicamente , Hipoglicemiantes , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucose , SódioRESUMO
A common practice exists in hospitals where extra tubes of blood are collected for possible add-on testing, this practice contributes to wastage of consumables. Baseline estimates from a 5-month local lab information system audit revealed that ~65 extra tubes per day were being collected, with an additional 2-week manual audit of all extra tubes received in the laboratory confirming the practice. The audits showed that the majority of the tubes (~99%) were being drawn from the adult emergency department (ED). Furthermore, only 5% of the extra tubes were being used for add-on testing, whereas the remaining tubes had no testing performed on them and were discarded at the end of the day. This translates to over 23 000 extra tubes being wasted annually.After initial discussion with ED leadership, the practice was identified as primarily nurse driven. An educational intervention was created and entitled 'Every Tube Counts', with the aim to reduce extra tube collections in the adult ED by 50% within the first month of intervention. First, a memo with initial findings and a request to stop the practice of extra tube collection was sent out to all ED staff. After 2 weeks of additional data collection, it was noticed that extra tubes were still being collected. A second intervention, which consisted of another communication and utilisation of nurse educators to disseminate the information to nursing staff, saw a remarkable ~80% reduction in collection of extra tubes in the following few months after the second intervention. The practice was followed for an additional 15 months, which saw a slight increase of extra tube collections over time with a levelling off towards the latter period of the study. However, the target goal was maintained over the entire study period.
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Serviço Hospitalar de Emergência , Hospitais , Adulto , Humanos , Comunicação , Coleta de DadosRESUMO
Microbiology sample swabs may be unsuccessful or rejected for a variety of reasons. Typically, errors occur in the preanalytical phase of sample collection. Errors with collection, handling and transport can lead to the need to repeat specimen collection. Unsuccessful specimens contribute to delays in diagnosis, increased patient stress and increased healthcare costs. An audit of sample swabs from London Health Sciences Centre Children's Hospital from August through October 2021 yielded complete success rates of 100% for ear and eye culture swabs, 98.1% for methicillin-resistant Staphylococcus aureus swabs and 88.9% for wound swabs. This project aimed to improve wound swab success to 95% on the paediatric inpatient and paediatric emergency departments by May 2022.Stakeholders from paediatric clinical services including physicians, nurses and the laboratory medicine team at our centre were engaged to guide quality improvement interventions to improve specimen success rate. Based on feedback, we implemented visual aids to our electronic laboratory test information guide. Additionally, visual reminders of correct sample collection equipment were placed in high traffic areas for nursing staff.After the interventions were implemented, a three-month follow-up showed that wound swab success rate rose to 95.3%. This study achieved its aim of improving wound swab success rate to 95%. It adds to the growing pool of evidence that preanalytical phase intervention such as visual aids can increase swab success rates, in healthcare settings.
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Staphylococcus aureus Resistente à Meticilina , Humanos , Criança , Melhoria de Qualidade , Pacientes Internados , Recursos Audiovisuais , Análise por ConglomeradosRESUMO
Cancers rely on multiple, heterogeneous processes at different scales, pertaining to many biomedical fields. Therefore, understanding cancer is necessarily an interdisciplinary task that requires placing specialised experimental and clinical research into a broader conceptual, theoretical, and methodological framework. Without such a framework, oncology will collect piecemeal results, with scant dialogue between the different scientific communities studying cancer. We argue that one important way forward in service of a more successful dialogue is through greater integration of applied sciences (experimental and clinical) with conceptual and theoretical approaches, informed by philosophical methods. By way of illustration, we explore six central themes: (i) the role of mutations in cancer; (ii) the clonal evolution of cancer cells; (iii) the relationship between cancer and multicellularity; (iv) the tumour microenvironment; (v) the immune system; and (vi) stem cells. In each case, we examine open questions in the scientific literature through a philosophical methodology and show the benefit of such a synergy for the scientific and medical understanding of cancer.
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Neoplasias , Filosofia , Pesquisa , Estudos InterdisciplinaresRESUMO
Extramedullary hematopoiesis (EMH) is a rare complication of solid tumor malignancies. We describe the first case of a patient who developed EMH in the pericardium secondary to metastatic gastrointestinal or pancreaticobiliary cancer. A 58-year-old man presented with recurrent episodes of fatigue and shortness of breath and was treated with thoracocentesis and pericardiocentesis for pleural and pericardial effusions, respectively. Owing to a markedly elevated alkaline phosphatase, a bone scan was performed and demonstrated diffuse sclerotic lesions. Evaluation of pleural effusion diagnosed metastatic adenocarcinoma, and cytospin morphology of the pericardial fluid demonstrated EMH. While EMH secondary to solid tumors is commonly suggested to be due to cytokine signaling, we propose the mechanism of EMH in this patient was due to extensive disruption of bone marrow hematopoiesis, similar to what is seen in myeloproliferative neoplasms.
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BACKGROUND: Erythrocytosis, most often measured as an increase in hemoglobin and/or hematocrit, is a common reason for referral to internal medicine and hematology clinics and a rational approach is required to effectively identify patients with polycythemia vera while avoiding over-investigation. AIM: We aimed to develop and validate a simple rule to predict JAK2 mutation positivity based on complete blood count parameters to aid in the diagnostic approach to patients referred for elevated hemoglobin. SETTING: Internal medicine and hematology clinics at an academic tertiary referral center. PARTICIPANTS: The JAK2 Prediction Cohort (JAKPOT), a large retrospective cohort (n = 901) of patients evaluated by internal medicine and hematology specialists for elevated hemoglobin. DESIGN: JAK2 mutation analysis was performed in all patients and clinical and laboratory variables were collected. Patients were randomly divided into derivation and validation cohorts. A prediction rule was developed using data from the derivation cohort and tested in the validation cohort. KEY RESULTS: The JAKPOT prediction rule included three variables: (i) red blood cell count >6.45×1012/L, (ii) platelets >350×109/L, and (iii) neutrophils >6.2×109/L; absence of all criteria was effective at ruling out JAK2-positivity with sensitivities 94.7% and 100%, and negative predictive values of 98.8% and 100% in the derivation and validation cohorts, respectively, with an overall low false negative rate of 0.4%. The rule was validated for three different methods of JAK2 testing. Applying this rule to our entire cohort would have resulted in over 50% fewer tests. CONCLUSION: In patients with elevated hemoglobin, the use of a simple prediction rule helps to accurately identify patients with a low likelihood of having a JAK2 mutation, potentially limiting costly over-investigation in this common referral population.
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Policitemia Vera , Policitemia , Humanos , Estudos Retrospectivos , Policitemia Vera/diagnóstico , Policitemia Vera/genética , Policitemia/genética , Hemoglobinas/genética , Mutação , Janus Quinase 2/genéticaRESUMO
BACKGROUND: Molecular testing for JAK2 mutations is part of the standard diagnostic workup for patients with suspected polycythemia vera. We sought to characterize evolving practice patterns in the investigation of erythrocytosis and the prevalence of secondary causes, including use of medications such as sodium-glucose cotransporter-2 (SGLT2) inhibitors, among patients who underwent molecular testing. METHODS: We reviewed charts of all consecutive patients investigated for erythrocytosis (hemoglobin > 160 g/L for women, > 165 g/L for men) with JAK2 testing between 2015 and 2021 at London Health Sciences Centre, a tertiary referral centre in Ontario, Canada, to assess changes in rates of JAK2 mutation positivity, average hemoglobin levels and the prevalence of secondary causes of erythrocytosis. RESULTS: A total of 891 patients with erythrocytosis underwent JAK2 mutation testing with an increase in number of tests (particularly from 2017 to 2018), a decrease in the rate of JAK2 positivity and similar average hemoglobin levels over the study period. We observed a high proportion of patients with secondary causes of erythrocytosis, ranging from 59% to 74% over the study period, including medications associated with erythrocytosis, namely testosterone (6%-11%) and SGLT2 inhibitors (2%-19%). Stopping SGLT2 inhibitors was associated with a significant decrease in hemoglobin levels (mean -14.7 g/L, 95% confidence interval -18.9 to -10.5 g/L) compared with continuation. INTERPRETATION: Use of SGLT2 inhibitors may be a common and underrecognized secondary cause of elevated hemoglobin levels in patients investigated for erythrocytosis. Our findings underscore the importance of a detailed medical history to support judicious use of molecular testing, in adherence with the current guideline on the investigation of erythrocytosis.
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Policitemia Vera , Policitemia , Inibidores do Transportador 2 de Sódio-Glicose , Masculino , Humanos , Feminino , Policitemia Vera/diagnóstico , Policitemia Vera/epidemiologia , Policitemia Vera/genética , Policitemia/diagnóstico , Policitemia/epidemiologia , Policitemia/genética , Ontário/epidemiologia , Técnicas de Diagnóstico Molecular , Hemoglobinas/genéticaRESUMO
INTRODUCTION: T-cell clonality testing by T-cell receptor (TCR) gene rearrangement is key to the diagnosis of T-cell lymphoproliferative disorders such as T-cell large granular lymphocytic (T-LGL) leukemia. Benign clonal T-cell expansions, however, are commonly found in patients without identifiable disease, a condition referred to as T-cell clones of uncertain significance (T-CUS). In practice, T-cell clonality testing is performed for a range of reasons and results are often challenging to interpret given the overlap between benign and malignant clonal T-cell proliferations and uncertainties in the management of T-CUS. METHODS: We conducted a 5-year retrospective cohort study of 211 consecutive patients who underwent PCR-based T-cell clonality testing for suspected T-LGL leukemia at our institution to characterize the use of T-cell clonality testing and its impact on patient management. RESULTS: Overall, 46.4% (n = 98) of individuals tested had a clonal T-cell population identified. Patients with a monoclonal T-cell population were more likely to be older, have rheumatoid arthritis and have higher lymphocyte counts compared to patients with polyclonal populations. The majority of patients eventually diagnosed and treated for T-LGL leukemia had rheumatoid arthritis and lower neutrophil counts compared to untreated patients with monoclonal T-cell populations. A diagnosis of T-LGL leukemia was made in only a minority of patients (n = 48, 22.7%), and only a small proportion were treated (n = 17, 8.1%). CONCLUSION: Our study suggests that T-cell clonality testing most commonly identifies incidental T-cell clones with only a minority of patients receiving a diagnosis of T-LGL leukemia and fewer requiring active treatment. These finding indicate an opportunity to improve utilization of T-cell clonality testing in clinical practice to better target patients where the results of testing would impact clinical management.
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Artrite Reumatoide , Leucemia Linfocítica Granular Grande , Humanos , Artrite Reumatoide/patologia , Células Clonais/patologia , Leucemia Linfocítica Granular Grande/diagnóstico , Leucemia Linfocítica Granular Grande/genética , Leucemia Linfocítica Granular Grande/patologia , Estudos Retrospectivos , Linfócitos T/patologiaRESUMO
Background: Since the identification of JAK2 V617F and exon 12 mutations as driver mutations in polycythemia vera (PV) in 2005, molecular testing of these mutations for patients with erythrocytosis has become a routine clinical practice. However, the incidence of myeloid mutations other than the common JAK2 V617F mutation in unselected patients referred for elevated hemoglobin is not well studied. This study aimed to characterize the mutational landscape in a real-world population of patients referred for erythrocytosis using a targeted next-generation sequencing (NGS)-based assay. Method: A total of 529 patients (hemoglobin levels >160 g/L in females or >165 g/L in males) were assessed between January 2018 and May 2021 for genetic variants using the Oncomine Myeloid Research Assay (ThermoFisher Scientific, Waltham, MA, USA) targeting 40 key genes with diagnostic and prognostic implications in hematological conditions (17 full genes and 23 genes with clinically relevant "hotspot" regions) and a panel of 29 fusion driver genes (>600 fusion partners). Results: JAK2 mutations were detected in 10.9% (58/529) of patients, with 57 patients positive for JAK2 V617F, while one patient had a JAK2 exon 12 mutation. Additional mutations were detected in 34.5% (20/58) of JAK2-positive patients: TET2 (11; 19%), DNMT3A (2;3.4%), ASXL1 (2; 3.4%), SRSF2 (2; 3.4%), BCOR (1; 1.7%), TP53 (1; 1.7%), and ZRSR2 (1; 1.7%). Diagnosis of PV was suspected in 2 JAK2-negative patients based on the 2016 World Health Organization (WHO) diagnostic criteria. Notably, one patient carried mutations in the SRSF2 and TET2 genes, and the other patient carried mutations in the SRSF2, IDH2, and ASXL1 genes. Three JAK2-negative patients with elevated hemoglobin who tested positive for BCR/ABL1 fusion were diagnosed with chronic myeloid leukemia (CML) and excluded from further analysis. The remaining 466 JAK2-negative patients were diagnosed with secondary erythrocytosis and mutations were found in 6% (28/466) of these cases. Conclusion: Mutations other than JAK2 mutations were frequently identified in patients referred for erythrocytosis, with mutations in the TET2, DNMT3A, and ASXL1 genes being detected in 34.5% of JAK2-positive PV patients. The presence of additional mutations, such as ASXL1 mutations, in this population has implications for prognosis. Both the incidence and mutation type identified in patients with secondary erythrocytosis likely reflects incidental, age-associated clonal hematopoiesis of indeterminate potential (CHIP).
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Policitemia Vera , Policitemia , Masculino , Feminino , Humanos , Policitemia Vera/genética , Mutação/genética , Sequenciamento de Nucleotídeos em Larga Escala , Hemoglobinas/genéticaRESUMO
BACKGROUND: The use of molecular genetic biomarkers is rapidly advancing to aid diagnosis, prognosis, and clinical management of hematological disorders. We have implemented a next-generation sequencing (NGS) assay for detection of genetic variants and fusions as a frontline test for patients suspected with myeloid malignancy. In this study, we summarize the findings and assess the clinical impact in the first 1613 patients tested. METHODS: All patients were assessed using NGS based Oncomine Myeloid Research Assay (ThermoFisher) including 40 genes (17 full genes and 23 genes with clinically relevant "hotspot" regions), along with a panel of 29 fusion driver genes (including over fusion 600 partners). RESULTS: Among 1613 patients with suspected myeloid malignancy, 43% patients harbored at least one clinically relevant variant: 91% (90/100) in acute myeloid leukemia patients, 71.7% (160/223) in myelodysplastic syndrome (MDS), 77.5% (308/397) in myeloproliferative neoplasm (MPN), 83% (34/41) in MPN/MDS, and 100% (40/40) in chronic myeloid leukemia patients. Comparison of NGS and cytogenetics results revealed a high degree of concordance in gene fusion detection. CONCLUSIONS: Our findings demonstrate clinical utility and feasibility of integrating a NGS-based gene mutation and fusion testing assay as a frontline diagnostic test in a large reported cohort of patients with suspected myeloid malignancy, in a clinical laboratory setting. Overlap with cytogenetic test results provides opportunity for testing reduction and streamlining.
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Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Transtornos Mieloproliferativos , DNA , Fusão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Mutação , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/genética , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/genética , RNAAssuntos
Eritropoetina , Policitemia , Eritropoetina/genética , Humanos , Janus Quinase 2/genética , Mutação , Policitemia/genéticaRESUMO
INTRODUCTION: In most laboratories, next generation sequencing (NGS) has been added without consideration for redundancy compared to conventional cytogenetics (CG). We tested a streamlined approach to genomic testing in patients with suspected myeloid and plasma cell neoplasms using next generation sequencing ("NGS first") as the primary testing modality and limiting cytogenetics (CG) to samples with morphologic abnormalities in the marrow aspirate. METHODS: Based on morphologic interpretation of bone marrow aspirate and flow cytometry, samples were triaged into four groups: (a) Samples with dysplasia or excess blasts had both NGS and karyotyping; (b) Samples without excess blasts or dysplasia had NGS only; (c) Repeat samples with previous NGS and/or CG studies were not retested; (d) Samples for suspected myeloma with less than 5% plasma cell had CG testing cancelled. RESULTS: Seven hundred eleven adult bone marrow (BM) samples met the study criteria. The NGS first algorithm eliminated CG testing in 229/303 (75.6%) of patients, primarily by reducing repeat testing. Potential cost avoided was approximately $124 000 per annum. Hematologists overruled the triage comment in only 11/303 (3.6%) cases requesting CG testing for a specific indication. CONCLUSIONS: Utilizing NGS as the primary genomic testing modality NGS was feasible and well accepted, reducing over three quarters of all CG requests and improving the financial case for adoption of NGS. Key factors for the success of this study were collaboration of clinical and genomic diagnostic teams in developing the algorithm, rapid turnaround time for BM interpretation for triage, and communication between laboratories.
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Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Adulto , Análise Citogenética , Citogenética , HumanosRESUMO
We have developed a rapid, accurate, and cost-effective serologic test for SARS-CoV-2 virus, which caused the COVID-19 pandemic, on the basis of antibody-dependent agglutination of antigen-coated latex particles. When validated using plasma samples that are positive or negative for SARS-CoV-2, the agglutination assay detected antibodies against the receptor-binding domain of the spike (S-RBD) or the nucleocapsid protein of SARS-CoV-2 with 100% specificity and â¼98% sensitivity. Furthermore, we found that the strength of the S-RBD antibody response measured by the agglutination assay correlated with the efficiency of the plasma in blocking RBD binding to the angiotensin-converting enzyme 2 in a surrogate neutralization assay, suggesting that the agglutination assay might be used to identify individuals with virus-neutralizing antibodies. Intriguingly, we found that >92% of patients had detectable antibodies on the day of a positive viral RNA test, suggesting that the agglutination antibody test might complement RNA testing for the diagnosis of SARS-CoV-2 infection.
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COVID-19 , SARS-CoV-2 , Humanos , Pandemias , COVID-19/diagnóstico , Anticorpos Antivirais , AglutinaçãoRESUMO
BACKGROUNDThe role of humoral immunity in COVID-19 is not fully understood, owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome.METHODSUsing SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients' plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution.RESULTSWe identified linear epitopes from the spike (S) and nucleocapsid (N) proteins and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S-811-825, S-881-895, and N-156-170 epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes.CONCLUSIONEpitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, but it also may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern in both the peptide array and latex agglutination formats.FUNDINGOntario Research Fund (ORF) COVID-19 Rapid Research Fund, Toronto COVID-19 Action Fund, Western University, Lawson Health Research Institute, London Health Sciences Foundation, and Academic Medical Organization of Southwestern Ontario (AMOSO) Innovation Fund.