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.

Oral estrogen leads to falsely low concentrations of estradiol in a common immunoassay

Objectives: Recently, an estradiol immunoassay manufacturer (Beckman Coulter, USA) issued an “Important Product Notice” alerting clinical laboratories their assay (Access Sensitive Estradiol) was not indicated for patients undergoing exogenous estradiol treatment. The objective of this analysis was to evaluate immunoassay bias relative to liquid chromatography tandem mass spectrometry (LC-MS/MS) in transgender women and to examine the influence of unconjugated estrone on measurements. Design: Cross sectional secondary analysis. Methods: Estradiol concentrations from 89 transgender women were determined by three immunoassays (Access Sensitive Estradiol [‘New BC’] and Access Estradiol assays [‘Old BC’], Beckman Coulter; Estradiol III assay [‘Roche’], Roche Diagnostics) and LC-MS/MS. Bias was evaluated with and without adjustment for estrone concentrations. The number of participants who shifted between three estradiol concentration ranges for each immunoassay versus LC-MS/MS (>300 pg/mL, 70-300 pg/mL, and <70 pg/mL) was calculated. Results: The New BC assay had the largest magnitude overall bias (median: -34%) and was -40%, -22%, and -10%, among participants receiving tablet, patch, or injection preparations, respectively. Overall bias was -12% and +17% for the Roche and Old BC assays, respectively. When measured with the New BC assay, 18 participants shifted to a lower estradiol concentration range (versus 9 and 10 participants based on Roche or Old BC assays, respectively). Adjustment for estrone did not minimize bias. Conclusions: Immunoassay measurement of estradiol in transgender women may lead to falsely decreased concentrations that have the potential to affect management. A multi-disciplinary health care approach is needed to ensure appropriate analytical methods are available.

Method comparison of three serum free light chain assays on the Roche Cobas 6000 c501 chemistry analyzer

Objectives Free light chains (FLC) are important in the diagnosis, prognosis and monitoring of therapy response of patients with monoclonal gammopathies. In this study, we performed a method comparison of three FLC assays on the Cobas 6000 c501 chemistry analyzer of Roche Diagnostics. Methods Samples of 119 patients with various monoclonal gammopathies and 26 control patients were measured with the Freelite (The Binding Site), Diazyme (Diazyme Laboratories) and KLoneus (Trimero Diagnostics) FLC assays. A method comparison was performed and reference intervals of the three assays were validated. Results The analysis of the Bland-Altman agreement showed bias between the three FLC assays, ranging from −62.7 to 5.1% for κFLC and between −29.2 to 80.5% for λFLC. The Freelite and Diazyme assays have the highest agreement. The concordance of the FLC-ratio ranges from 41 to 75%, with the highest concordance between the Freelite and KLoneus assays. The FLC-ratio in 25 sera from healthy controls were within the reference ranges of the Freelite and KLoneus assays. The FLC-ratio was elevated in all 25 samples tested with the Diazyme assay. Conclusions The agreement for the free light chains is highest between the Freelite and the Diazyme assay and fair for the KLoneus assay. However, concordance of the FLC-ratio is highest when the Freelite and KLoneus assays were compared. Our data suggest that concordance for the Diazyme assay could be improved by recalibration. Because of absolute differences between the three methods in individual patients, none of the three FLC assays can be used interchangeably.

КЛІНІКО-ЛАБОРАТОРНА ХАРАКТЕРИСТИКА КОМОРБІДНОГО ПЕРЕБІГУ ЦУКРОВОГО ДІАБЕТУ 2 ТИПУ З НАДМІРНОЮ МАСОЮ ТІЛА/ОЖИРІННЯМ ТА АРТЕРІАЛЬНОЮ ГІПЕРТЕНЗІЄЮ

Вступ. Артеріальна гіпертензія (АГ) є основною причиною захворюваності та смертності серед пацієнтів із цукровим діабетом 2 типу (ЦД2), що пов’язано із серцево-судинними захворюваннями. Мета дослідження – проаналізувати основні клініко-лабораторні фактори коморбідного перебігу цукрового діабету 2 типу з надмірною масою тіла/ожирінням та артеріальною гіпертензією. Методи дослідження. Ретроспективний аналіз медичної документації проведено в 579 хворих на цукровий діабет 2 типу (T2DM). Концентрацію глюкози та інших біохімічних показників визначали з використанням стандартного набору на автоматичному біохімічному аналізаторі “COBAS INTEGRA® 400” (“Roche Diagnostics”). Показники загального аналізу крові визначали на автоматичному гематологічному аналізаторі “Yumizen H500 CT”. Результати й обговорення. При проведенні аналізу рангових варіацій Краскела – Уолліса не виявлено цінності показників загального аналізу крові та загального аналізу сечі в діагностиці коморбідного перебігу ЦД2, надмірної маси тіла/ожиріння та АГ. Під час аналізу біохімічних параметрів встановлено найвищі рівні сечовини і сечової кислоти у хворих на ЦД2, ожиріння та АГ. Аналіз показників вуглеводного обміну показав, що рівні глюкози, інсуліну та індекс HOMA у крові пацієнтів різних дослідних груп вірогідно різнилися при проведенні аналізу рангових варіацій Краскела – Уолліса. Висновки. Під час аналізу статевого диморфізму встановлено переважання осіб жіночої статі з коморбідним перебігом цукрового діабету 2 типу з надмірною масю тіла/ожирінням та артеріальною гіпертензією. При цьому серед хворих на цукровий діабет 2 типу з коморбідною артеріальною гіпертензією виявлено вірогідно вищий відсоток хворих чоловіків з нормальною масою тіла. При аналізі антропомет­ричних показників пацієнтів різних груп встановлено, що у пацієнтів 6-ї групи зріст був вірогідно меншим стосовно даних у хворих на цукровий діабет 2 типу з нормальною масою тіла та при поєднанні з артеріальною гіпертензією. Індекс маси тіла був достовірно вищим у пацієнтів з надмірною масою тіла/ожирінням. У хворих з коморбідним перебігом цукрового діабету 2 типу з надмірною масою тіла/ожирінням та артеріальною гіпертензією встановлено вірогідно вищі рівні сечовини (на 24,4 %) та сечової кислоти (на 93,0 %) щодо даних у пацієнтів із ЦД2 та нормальною масою тіла.

Establishment of a quantitative RT-PCR detection of SARS-CoV-2 virus

Background The outbreak of novel coronavirus disease 2019 (COVID-19) has become a public health emergency of international concern. Quantitative testing of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus is demanded in evaluating the efficacy of antiviral drugs and vaccines and RT-PCR can be widely deployed in the clinical assay of viral loads. Here, we developed a quantitative RT-PCR method for SARS-CoV-2 virus detection in this study. Methods RT-PCR kits targeting E (envelope) gene, N (nucleocapsid) gene and RdRP (RNA-dependent RNA polymerase) gene of SARS-CoV-2 from Roche Diagnostics were evaluated and E gene kit was employed for quantitative detection of COVID-19 virus using Cobas Z480. Viral load was calculated according to the standard curve established by series dilution of an E-gene RNA standard provided by Tib-Molbiol (a division of Roche Diagnostics). Assay performance was evaluated. Results The performance of the assay is acceptable with limit of detection (LOD) below 10E1 copies/μL and lower limit of quantification (LLOQ) as 10E2 copies/μL. Conclusion A quantitative detection of the COVID-19 virus based on RT-PCR was established.

suPAR cut-offs for stratification of low, medium, and high-risk acute medical patients in the emergency department

Background Soluble urokinase plasminogen activator receptor (suPAR) levels have previously been associated with readmission and mortality in acute medical patients in the ED. However, no specific cut-offs for suPAR have been tested in this population. Methods Prospective observational study of consecutively included acute medical patients. Follow-up of mortality and readmission was carried out for 30- and 90 days stratified into baseline suPAR < 4, 4–6 and > 6 ng/ml. suPAR levels were measured using suPARnostic® Turbilatex assay on a Cobas c501 (Roche Diagnostics Ltd) analyser. Results A total of 1747 acute medical patients in the ED were included. Median age was 70 (IQR: 57–79) and 51.4% were men. Adjusted linear regression analysis showed that suPAR, independently of age, sex and C-reactive protein levels, predicted 30- and 90-day mortality (Odds ratio for doubling in suPAR 1.96 (95% confidence intervals: 1.42–2.70) Among patients with suPAR below 4 ng/ml (N = 804, 46.0%), 8 (1.0%) died within 90-day follow-up, resulting in a negative predictive value of 99.0% and a sensitivity of 94.6%. Altogether 514 (29.4%) patients had suPAR of 4–6 ng/ml, of whom 43 (8.4%) died during 90-day follow-up. Among patients with suPAR above 6 ng/ml (N = 429, 24.6%), 87 patients (20.3%) died within 90-day follow-up, resulting in a positive predictive value of 20.1% and a specificity of 78.7%. Conclusions suPAR cut-offs of below 4, between 4 and 6 and above 6 ng/ml can identify acute medical patients who have low, medium or high risk of 30- and 90-day mortality. The turbidimetric assay provides suPAR results within 30 min that may aid in the decision of discharge or admission of acute medical patients.

BNT162b2 mRNA COVID-19 Vaccination (BNTCV) in Patients (Pts) Having Hematological Malignancies (HM): Early tolerance with telemedicine application connecting and antibody anti-Spike IgG response (AcAS)

Pts with HM and particularly those under therapy or with secondary immune deficiency have been reported to have a low or delayed specific immune response. In addition, pts receiving rituximab for either HM or auto-immune disease presented a low specific antibody response. Daratumumab, an anti-CD38 moAb was demonstrated to lower normal plasma cells and to reduce polyclonal IgA, M and E in pts with multiple myeloma (MM). It is fundamental to evaluate the early post-vaccination tolerance and the level of seroconversion for such pts. Patients' population. 194 pts having HM and followed at the Institute (IC), received at least 2 doses of BNTCV (BioNTech Pfizer, Paris, France) by IM route with 3 weeks between the 2 doses. Currently, analysis was performed on 147 pts, including 63 pts with non-Hodgkin lymphoma (NHL), 18 with B-chronic lymphocytic leukemia (CLL), 34 with MM, 14 with monoclonal gammopathy of undetermined significance (MGUS) and 18 with myeloproliferative disorder (MPD). Mean age was 69 years-old (range 27-92). Follow-up of early tolerance in pts using a telemedicine application. 43 pts vaccinated at the IC received Thess®, a telemedicine system connecting the patient to the IC, developed by La Valeriane Inc. (Montpellier, France, www.thess-corp.fr) 24/24h, 7days. Local pain (&lt;1 day) was common and transient, particularly reported after the 2nd dose. Only 4/43 patients reported significant adverse events through telemedicine and followed by a medical call, including mainly severe asthenia for 2 days or more, fever (&gt;38°C) for at least 2 days, headache, or general pain. The satisfaction survey of monitoring system was good. In addition, adherence to vaccination was excellent with only one refusal out of the 194 pts. AcAS follow-up IgG AcAS and IgG+M AcAS were analyzed in the serum 3 to 4 weeks after the 1st dose and 4-8 weeks after the 2 nd dose and every 2 months, respectively by SARS-CoV-2 IgG II Quant ® Assay with a threshold of positivity at 50 AU/mL (Abbott, Rungis, France) and Elecsys ® Anti-SARS-CoV-2 S (Roche Diagnostics, Meylan, France) with a threshold of positivity at 0.8 U/mL. 270 samples were analyzed in duplicate with the 2 assays, by 2 independent labs. Data were reported and statistically analyzed by the clinical research team. 17 results were discordant including 12 with Abbott IgG test undetectable and Roche IgG+M detectable, and 5 with Abbott test detectable and Roche undetectable. After the first dose of BNTCV, 72/147 (49%) pts were seroconverted, including 7/18 (39%) with CLL, 27/63 (43%) with NHL, 10/14 (71%) with MGUS, 17/34 (50%) with MM and 11/18 (61%) with SMD. The median levels of the AcAS response for pts were 0 (range 0-40 000 AU/mL) for the AdviseDX test as compared to 12 normal subjects (mean 257 AU/mL, range 226-283), and 0 U/mL (range 0-2500) for the Elecsys test. 49/75 (65%) of untreated pts were seroconverted after the 1 st dose as compared to 26/72 (36%) of treated patients (p&lt;0.001), with rituximab or ibrutinib as negative factors in addition to low gammaglobulin level (&lt;5g/L, p=0.019), similarly to the IgG level. Analysis of CRP levels, circulating lymphocyte counts, and lymphocyte subpopulations will be performed. After the second dose of BNTCV, 50 pts have currently been tested, with only 25 additional pts seroconverted. The median levels of the AcAS response for pts were 310 AU/mL (range 62-11760) for the AdviseDX test as compared to 12 normal subjects (mean 6725 AU/mL, range 3002-9787), and 1250 U/mL (range 0.87-2500) for the Elecsys test. The 25 patients who were not seroconverted after the 2nd dose received a 3rd dose of BNTCV, including 8 with MM, 9 with NHL, 5 with CLL, 1 with MGUS (with polyneuropathy under daratumumab) and 2 with SMD. Currently, 9 pts were tested after the 3 rd dose. AcAS levels were respectively 268 AU/mL for 1 MM treated by daratumumab, 103 and 313 AU/mL for 2 MM under carfilzomib, 1622 AU/mL for 1 untreated SMD, 29100 AU/mL for 1 untreated MGUS, 537 AU/mL for 1 CLL and 2 negative NHL. In conclusion, there is a need to follow AcAS for pts having HM including SMD after BNTCV aiming to adapt vaccine strategy including a 3 rd dose and eventual recall. As some pts are always negative after a 3 rd dose, vaccination strategy could be discussed by using different combinations of vaccine or the addition of immune adjuvant in a more personalized medicine. In addition, the usage of telemedicine connecting system may help to follow the early tolerance and to improve the pts' adherence. Disclosures Rossi: NPO Petrovax Pharm: Consultancy; LEO Pharma: Consultancy; EUSA Pharma: Consultancy; E-SANA Inc: Other: Co-founder of E-SANA Inc .

Humoral Response after Vaccination with Half-Dose of BNT162b2 in Subjects under 55 Years of Age

In the context of the ongoing COVID-19 pandemic, using a half-dose schedule vaccination can help to return to normalcy in a cost-efficient manner, which is especially important for low and middle-income countries. We undertook a study to confirm that in adults up to 55 years old, the humoral response to the half-dose (15 µg, 35 participants between 18 and 55 years old) and to the recommended dose (30 µg, 155 participants) in the two-dose three-week interval schedule would be comparable. Antibody levels were measured by the Elecsys Anti-SARS-CoV-2 S assay (Roche Diagnostics, upper detection limit: 2570 BAU/mL) on the day of dose 2 of the vaccine and then 8–10 days later to assess peak response to dose 2. The difference in proportions between the participants who did and did not exceed the upper detection limit 8–10 days after dose 2 was not statistically significant (p = 0.152). We suggest that a half-dose schedule can help to achieve widespread vaccination coverage more quickly and cheaply.

Chất lượng bộ sinh phẩm Polyvac - SARS-CoV-2 IgG/M ELISA chẩn đoán phát hiện kháng thể SARS - COV-2 trên các mẫu lâm sàng

Nghiên cứu này nhằm mục tiêu đánh giá bộ sinh phẩm phát hiện kháng thể IgG/M kháng SARS-CoV-2 bằng ELISA do POLYVAC sản xuất (Polyvac SARS-CoV-2 IgG/M ELISA). Nghiên cứu được tiến hành trên 633 mẫu huyết thanh gồm 302 mẫu dương tính và 331 mẫu âm tính lấy từ bệnh nhân (2020) đã được khẳng định bằng RT-PCR. Có 200 mẫu dương tính và 200 mẫu âm tính được thực hiện so sánh với Elecsys Anti-SARS-CoV-2 của Roche Diagnostics. Kết quả cho thấy Polyvac SARS-CoV-2 IgG ELISA có độ nhạy 86,42%, độ đặc hiệu 100%; Polyvac SARS-CoV-2 IgM ELISA có độ nhạy 85,43% và độ đặc hiệu 95,77% so với RT-PCR trên các mẫu lâm sàng. Ở thời điểm sau khi có triệu chứng 0 - 7 ngày, 8 - 14 ngày, >14 ngày độ nhạy của bộ sinh phẩm tương ứng trung bình là 34,15 % ( 21,56% - 44,45%), 92,05% (84,49% - 96,17%), 97,97% (94,21% - 99,31) với IgG và 70,73% (55,22% - 82,39%), 78,41% (68,72% - 85,72%), 92,57% (87,18% - 95,80%) với IgM. Độ tương đồng giữa Polyvac SARS-CoV-2 IgG/M ELISA và Elecsys Anti-SARS-CoV-2 với mẫu âm tính là 81% (76,49 - 86,09 %), mẫu dương tính là 98,04% (94,40 - 99,33%), độ tương đồng giữa 2 bộ sinh phẩm là 88,04% (84,45-90,83%). Polyvac SARS-CoV-2 IgG/M ELISA có thể sử dụng trong giám sát dịch tễ huyết thanh học để xác định tiền sử có phơi nhiễm với SARS-CoV-2 và hỗ trợ chẩn đoán ở giai đoạn sau 7 ngày phát hiện có triệu chứng.