key: cord-0759399-e5wkc0jk
authors: Lau, C.S.; Hoo, S.P.; Koh, J.M.J.; Phua, S.K.; Aw, T.C.
title: PERFORMANCE OF THE ROCHE IL-6 CHEMILUMINESCENT IMMUNOASSAY IN PATIENTS WITH COVID-LIKE RESPIRATORY SYMPTOMS
date: 2021-06-29
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2021.114224
sha: dac4409ef169e03f3270a5b78ca5365b4832f698
doc_id: 759399
cord_uid: e5wkc0jk

INTRODUCTION: We evaluated the Roche Elecsys IL6 assay on the Cobas immunoassay analyser. METHOD: Serum IL6 of 144 controls were compared to 52 samples from patients with COVID-like respiratory symptoms (17 SARS-CoV-2 RT-PCR positive); 25 of these were from the intensive care unit (ICU). We compared the IL6 levels to C-reactive protein (CRP) and procalcitonin (PCT) levels in all cases. RESULTS: The IL6 assay had coefficient-of-variation (CV) of 2.3% (34.1 pg/mL) and 2.5% (222.5 pg/mL), a limit of quantitation <1.6 pg/mL, and was linear from 1.6-4948pg/mL. There was a significant difference in IL6 values between patients with COVID-like respiratory symptoms versus controls (p < 0.001). ROC analysis showed that IL6 > 6.4 pg/mL identified symptomatic cases (AUC 0.94, sensitivity 88.2%, specificity 97.2%). There was a significant difference between the IL6 of symptomatic ICU/non-ICU cases (median IL6 228 vs 11 pg/mL, p < 0.0001); ROC analysis showed IL6 > 75 pg/mL (sensitivity 76.0%, specificity 88.9%) was superior to CRP and PCT in predicting ICU admission (AUC: IL6 0.83, CRP 0.71, PCT 0.82). CONCLUSION: The performance of Elecsys IL6 assay is in keeping with the manufacturer’s claims. IL6 > 6.4 pg/mL differentiates healthy from suspected COVID-19 cases and appears to be raised earlier than the other inflammatory markers in some cases. IL6 > 75 pg/mL was a good predictor of ICU admission.

IL6 induces foam cell formation, the release of further inflammatory cytokines, and chemotaxis [1] . IL6 is elevated in patients with Coronavirus disease 2019 (COVID-19) related cytokine storm syndrome [2] . Although peak IL6 concentrations in COVID-19 infection are not as raised as in sepsis [3] , IL6 can reach up to 430pg/mL in cases of severe COVID-19 [4] , and in a study of patients with severe COVID-19 infection requiring intensive care unit (ICU) admission, IL6 was elevated up to 1000-fold compared to healthy controls [5] . Other studies also support the fact that IL6 is significantly elevated in severe COVID-19 requiring ICU admission [6] , and some studies [7] have used IL6 to predict survival outcomes in novel severe acute respiratory J o u r n a l P r e -p r o o f syndrome coronavirus 2 (SARS-CoV-2) infection. As such, the US Food and Drug administration has recently approved the use of several IL6 assays, for example, the Siemens IL6 assay [8] . The Roche Elecsys IL6 assay was approved for use by the US Food and Drug administration in June 2020 [9] (under emergency use authorization). We describe our evaluation of the Elecsys IL6 assay run on the Cobas e801 immunoassay analyser and compared the IL6 to C-reactive protein (CRP) and procalcitonin (PCT) in subjects with respiratory symptoms suspicious of COVID-19.

: 52 subjects with respiratory symptoms suspicious of COVID-19 (e.g. pneumonia, cough, fever) and tested with RT-PCR from April-June 2020 were recruited as cases (18 females, 34 males), using residual de-identified sera from other routine laboratory testing (e.g., renal panels). 17 of these cases were SARS-CoV-2 RT-PCR positive and 25 cases were from the ICU (9 RT-PCR positive, 16 RT-PCR negative). Samples from 144 (116 females, 28 males) healthy healthcare workers (HCWs) served as controls. As this work involved de-identified leftover sera and was part of evaluating a new diagnostic assay, it was deemed exempt by our institutional review board.

The Roche Elecsys IL6 assay is a non-competitive (sandwich) chemiluminescent immunoassay. 18uL of sample undergoes a first incubation with IL6 specific antibodies, followed by a second incubation with IL6 specific antibodies labelled with ruthenium complexes to form a sandwich complex. Thereafter, complexes are magnetically captured, where a voltage then induces a chemiluminescent emission directly proportional to the IL6 concentration. The assay has a claimed measuring range of 1.5-5000pg/mL, a limit of quantitation (LOQ) of 2.5pg/mL, an inter-assay precision (CV) of 17.4% (at 1.82pg/mL) and 2.0% (at 4461pg/mL). The stated reference interval is <7pg/mL. For RT-PCR testing, our hospital molecular laboratory employs a duplex real-time RT-PCR that targets the N and E J o u r n a l P r e -p r o o f genes using a Qiagen EZ1 extraction system and Rotor Gene Q amplification system. The Elecsys CRP assay is a particle enhanced immunoturbidimetric assay where CRP agglutinates with latex particles coated with monoclonal anti-CRP antibodies; the reference interval is <5mg/L. The Elecsys BRAHMS PCT assay is a non-competitive chemiluminescent immunoassay where PCT is incubated with monoclonal PCT-specific antibodies and PCTspecific antibodies labelled with a ruthenium complex, bound to solid phase; the reference interval for PCT is <0.5ng/mL.

For precision analysis, negative and positive Roche control materials were run 5 times each day for 5 days, as per the CLSI EP15-A3 protocol [10] . Assay linearity was assessed following the CLSI EP-6 protocol [11] using unidentified patient sera run in duplicates for different levels.

The limit of quantitation (LOQ) was verified with samples of pooled patient sera.

Statistical analyses were performed using MedCalc ® Statistical Software version 19.5.3 (MedCalc Software Ltd, Ostend, Belgium). We compared IL6 values between controls and cases with respiratory symptoms suspicious of COVID-19 (SARS-CoV-

The Elecsys IL6 assay had a CV of 2.3% (34.1pg/mL)/2.5% (222.5pg/mL). The assay was linear from 1.6-4948pg/mL. The LOQ was deemed to be 1.6pg/mL, as CV at this level was still 4.9% (95% CI 2.5-7.3%). This is lower than the manufacturer stated LOQ of 2.5pg/mL. Assay time was 18 minutes and results were available 1 minute later; throughput for the analysis of 50 samples was 29 minutes. For computation of results, values <1.6pg/mL are taken as 1.6pg/mL, and values reported as >5000pg/mL are taken as 5000pg/mL.

We compared the IL6 values between 3 groups: controls, SARS-CoV-2 RT-PCR negative cases with COVID-like respiratory symptoms, and cases positive for SARS-CoV-2 RT-PCR. There was a significant difference (p<0.0001) between the median IL6 values of the patients with suspected COVID-19 (COVIDlike respiratory symptoms with and without positive RT-PCR) and the controls (43.3 vs 2.3pg/mL), with a difference of 40.4pg/mL (see Figure 1a ). There was a wide spread of IL6 values between the RT-PCR negative (median 87.2pg/mL, IQR 7.0 to 63.5pg/mL) and RT-PCR positive subjects (median 12.7pg/mL, IQR 11.1 to 277.0pg/mL); the apparent difference between these 2 groups failed to achieve statistical significance (p=0.052) (see Figure 1b ). We performed ROC analysis between the IL6 values of the 144 healthy controls and the 52 cases with COVID-like respiratory symptoms. ROC analysis showed that an associated criterion of >6.4pg/mL could be used to separate cases and controls with a sensitivity of 88.5%

and specificity of 97.2% (AUC 0.96, p<0.001) (see Figure 2 ). We also compared the IL6, CRP and PCT values between ICU cases and non-ICU cases.

There was a significant difference between the IL6 values of both groups (median IL6 228 vs 11pg/mL, p<0.0001) (see Figure 3 ). Similarly, CRP and PCT was also significantly different in these groups (CRP 338 vs 210mg/L, p=0.009; PCT 1.49 vs 0.13ng/mL, p=0.0001 respectively). 

Studies [3] have shown that the estimated pooled mean IL6 in COVID-19 was 37pg/mL. Based on this cut-off, we stratified the IL6 values in our population. When cases were grouped based on initial IL6 levels (<6.4pg/mL, 6.4-37pg/mL, >37pg/mL), CRP and PCT generally increased in tandem with IL6 (see Table 1 ). 8 out of 52 patients had a normal normal CRP and PCT; IL6 was >6.4pg/mL in 5 of them. Two of them had IL6 of 5000pg/mL and 2088pg/mL (CRP 3.9 and 2.9mg/mL, PCT 0.05 and 0.25ng/mL respectively). Notably, all 6 cases in our study with IL6 >1000pg/mL (2088-5000pg/mL) were all ICU cases but were SARS-CoV-2 RT-PCR negative. levels between SARS-CoV-2 RT-PCR positive and negative cases with COVID-like respiratory symptoms. Part of the issue is how great variation exists between the performance of RT-PCR assays [14] . When the College of American Pathologists surveyed more than 700 laboratories with control materials, the median cycle threshold values reported for different methods varied by as much as 14 cycles [15] , and even within the same instrument, the difference in median cycle threshold values for different targets was as high as 3.0 cycles.

This may have contributed to the lack of distinction in IL6 values between RT-PCR negative and positive cases in our study. Secondly, IL6 is not specific to COVID-19 and can be raised in any cause of ARDS and sepsis [3] and in many disorders with chronic inflammation (e.g., rheumatoid arthritis) [16] . In addition, our COVID-19 caseload in this study was modest (n=17).

Despite this, IL6 still has use in patients suspicious for COVID-19. As shown in our study, the elevation of IL6 precedes that of CRP and PCT in some of our patients (9.6%, 5 out of 52 cases [19] showed that IL6 was better able to discriminate between sepsis and controls (AUC 0.89) while the AUC of CRP and PCT were 0.77 and 0.80 within 6 hours of the diagnosis of sepsis. The cumulative evidence suggests that IL6 is an earlier biomarker of severe infections than CRP or PCT.

IL6 of >75pg/mL was also superior to CRP and PCT in the prediction of ICU admission, and there was a significant difference in IL6 between ICU and non-ICU cases. This is supported by other studies as well. In one study [4] , maximal IL6 levels before intubation was the best predictor for mechanical ventilation, with an AUC of 0.97 compared to an AUC of 0.86 for CRP; presentation IL6 levels >35pg/mL had a high sensitivity (84%) for respiratory failure. The same study [4] also showed that IL6 levels could predict respiratory failure earlier than CRP [27] . In yet another study of 64 patients, the use of TCZ in earlier stages of disease resulted in an early favourable response responders to TCZ, with a recommended cut-off value of 177pg/mL to differentiate between the two groups. A recent study comparing TCZ treatment against a placebo in 389 COVID-19

patients [29] showed that although TCZ reduced the likelihood of disease progression (12% with TCZ vs 19% without TCZ), it did not improve survival (mortality 11.6% with TCZ and 11.8% without TCZ). The conflicting evidence of IL6-blockade in COVID-19 treatment has led to declining enthusiasm for its use as a treatment regimen and hence utilization of IL6. Indeed, in the latest guideline by the US National Institutes of Health [30] , the panel had no recommendation for or against the use of TCZ in the treatment of COVID-19 for patients who were within 24 hours of admission to the ICU requiring respiratory support. Although we did not have access to the medication history of the subjects in this study, TCZ use for COVID-19 treatment is approved for clinical trials only in our country, and our centre was not a study centre for TCZ treatment. We understand that none of the symptomatic cases in our study received TCZ treatment.

J o u r n a l P r e -p r o o f

The Roche Elecsys IL6 assay performs as claimed by the manufacturer but we found that the LoQ is lower. There is a clear difference between the IL6 of cases with respiratory symptoms suspicious of COVID-19 and healthy controls. IL6 may rise earlier than CRP and PCT in some cases with COVID-like respiratory symptoms, and IL6 is a good predictor for ICU admission in symptomatic cases.

All co-authors have contributed to the study and manuscript.

None.

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