Diagnostic accuracy and reliability remain key in SARS-CoV-2 testing

The SARS-CoV-2 strain of coronavirus, which leads to the disease COVID-19, presents in many as a minor cold or flu; however, for those with health complications, including autoimmune diseases, asthma, heart disease and diabetes, the risk of developing serious illness and adverse outcomes is much greater.

Currently, it is estimated that as many as 1 in 6 will experience complications that could be life-threatening.¹ Because of the spread and devastation of COVID-19, laboratory diagnostics plays an even more essential role in the diagnosis and management of suspected cases or affected patients. As in the past, clinicians are looking to accurate and reliable controls and reagents to provide the most efficacy in results.

As the spread and devastation of the COVID-19 pandemic continues to grow, it is vital that fast and accurate diagnostic testing strategies are implemented for effective risk stratification, monitoring of treatment and recovery.

Cytokine storms

A cytokine storm, a hyperactive immune response, is a grave complication associated with SARS-CoV-2, triggering life-threatening pneumonia, acute respiratory distress syndrome (ARDS) and multiple organ failure.^{2, 3} Early detection of such inflammatory markers can indicate the presence of a cytokine storm and allow timely therapeutic intervention.⁴

It is estimated that cytokine storms occur in up to 5 percent of severe COVID-19 cases, with high levels of several inflammatory cytokines including IL-6, IL-8, IL-10, and TNF-alpha identified. Due to the elevation of several pro-inflammatory and anti-inflammatory cytokines, a multiplex immunoassay approach can offer several advantages over the widely utilized single ELISA tests. The simultaneous detection of multiple cytokines from a single patient sample will provide clinicians with a detailed picture and complete patient profile, facilitating a personalized medicine approach.^{5, 6}

Renal function

The National Institute for Health and Care Excellence (NICE) recommends all COVID-19 patients are assessed for Acute Kidney Injury (AKI) on admission to a hospital and their condition monitored throughout their stay. AKI is a common complication of COVID-19, especially in diabetic patients.⁷

Serum creatinine (SCr) is the commonly utilized screening test for renal impairment; however, it is important to consider the accuracy and reliability of the method. The Jaffe and enzymatic methods are the readily available methods of SCr determination; and while the Jaffe method is less expensive, it is more susceptible to interferences. These interferences can lead to the misdiagnosis of patients, which isn’t ideal in the current pandemic.7 Moreover, the sensitivity of SCr in the early detection of renal disease is poor, as SCr is insensitive to small changes in glomerular filtration rate (GFR), with up to 50 percent of renal function potentially lost before significant SCr levels become detectable.^{8, 9} Cystatin C (CysC) is a superior marker of renal function and has been identified to be useful in the determination of the extent of renal damage, as well as distinguishing those with severe and mild COVID-19.¹⁰

Although CysC C is a superior marker of renal impairment, employing a multi-marker approach could identify renal disease or injury at a much earlier stage. Using current technologies, renal disease is typically diagnosed at around stage 4 or 5 when moderate to severe damage has already occurred. Using a multiplex approach, damage can be identified much earlier and in many cases before symptoms arise.

Hepatic function

Patients with abnormal liver function tests are at a significantly higher risk of developing severe disease, and complications such as pneumonia.11 Significantly elevated bilirubin levels, three times the upper limit, have been observed in COVID-19 patients.^{11, 12}

The diazo method is commonly utilized in bilirubin testing; however, superior methods exist. The vanadate oxidation (VO) method has many advantages particularly in hemolytic and lipemic samples. These advantages are particularly evident in neonatal and infant populations where hemolysis is extremely common. Moreover, the VO method offers a wider analytical measurement range.¹³

Other liver function markers are known to be elevated in COVID-19 patients including both AST and ALT, with markers like albumin decreased.

The importance of Lp(a) testing

Lipoprotein(a) / Lp(a) is a strong independent marker of coronary heart disease risk in patients with heterozygous familial hypercholesterolemia (HeFH) and has recently been identified as a key risk marker of cardiovascular complications in COVID-19 patients. Those with either baseline elevated Lp(a) or those whose Lp(a) levels increased following infection from COVID-19, or both, may be at a significantly increased risk of developing thromboses. Consideration should be given to measurement of Lp(a) and prophylactic anticoagulation of infected patients to reduce the risk. Elevated Lp(a) levels may also cause acute destabilization of pre-existing but quiescent, atherosclerotic plaques, which could induce an acute myocardial infarction or stroke.¹⁴

The biggest challenge that exists surrounding Lp(a) measurement is the heterogeneity of the apo(a) isoforms, resulting in the underestimation or overestimation of Lp(a) concentrations. In immunoassays, the variable numbers of repeated KIV-2 units in Lp(a) act as multiple epitopes. This is where standardization across calibrators is vital. Unless the calibrants do have the same range of isoforms as test samples, those with higher numbers of the KIV-2 repeat will represent with an overestimation in Lp(a) concentrations, while those with smaller numbers of the KIV-2 repeat will represent with an underestimation. The smaller isoforms are strongly associated with higher Lp(a) concentrations.¹⁵

The gold standard Lp(a) method is the Northwest Lipid Metabolism and Diabetes Research Laboratory (NLMDRKL) method, which employs an isoform insensitive antibody and is meticulously calibrated with well-characterized material; however, this test is not commercially available.¹⁵

With Lp(a) assays that are standardized to the WHO/IFCC (World Health Organization/International Federation of Clinical Chemistry) reference material, transferring values from mg/dl to nmol/l are more uniform. The assay considered the most reliable commercially available Lp(a) assay is so because:¹⁵

The isoform size variations are reduced as a range of calibrators from separate pools of serum used, which covered a range of Lp(a) concentrations.

The isoform size and concentrations are inversely correlated – better matching calibrants with test samples.

Methods are calibrated in nmol/l and traceable to WHO/IFCC reference material and give acceptable bias compared with the NLMDRL gold standard method.

Arguably as important as laboratory testing is the issue of internal and external quality control (QC). The accuracy and reliability of lab data and test results are looked to as the basis for most medical decisions. As such, it is even more imperative that the controls used in lab tests are of the highest quality.

Importance of internal QC

Seventy percent of medical decisions are based on laboratory data. Consequently, it is imperative that the information generated by the laboratory is accurate, timely and readily understandable by the end user. Quality control (QC) is run alongside the patient sample to determine the validity of the assay in accordance with the specifications.¹⁶ Consequently, QC ensures both accuracy and precision of patient sample results. QC should be designed to be commutable, so that the controls react to the test system in the same manner as the patient sample, which aids laboratories in meeting the ISO 15189:2012 requirement and ultimately ensures accurate and reliable instrument performance.17

ISO 15189:2012 states: “The laboratory should choose concentrations of control materials, wherever possible especially at or near clinical decisions values, which ensure the validity of decisions made.” Furthermore, ISO 15189:2012 also states: “Use of independent third-party control materials should be considered, either instead of, or in addition to, any control materials supplied by the reagent or instrument manufacturer.”

These requirements highlight that third-party controls should be employed to challenge laboratory instruments throughout the patient reportable range. The presence of analytes at clinically relevant decision levels not only aid in ensuring accurate instrument performance but maximize laboratory efficiency through the elimination of additional low/high concentration controls, an unnecessary additional expense.¹⁸

During the SARS-CoV-2 pandemic, it is vital that laboratories are adhering to and meeting the ISO 15189:2012 requirement to ensure accurate and reliable test results for COVID-19 adverse outcomes for swift and effective treatment plan implementation. In turn, this will ensure increasing confidence that performance mirrors that of the patient sample, producing accurate and reliable results, enabling effective treatment plan intervention.

Importance of external QC

External quality assessment (EQA) is a critical aspect of the laboratory quality management system. EQA is a method that enables a laboratory to compare their testing with another laboratory. The ISO/IEC Guide 43-1: 1997 states: “Proficiency testing schemes (PTS) are interlaboratory comparisons that are organized regularly to assess the performance of analytical laboratories and the competence of the analytical personnel.” EQA participation is essential for laboratories that are accredited, or plan to seek accreditation.¹⁹

ISO 15189:2012 addresses EQA requirements for laboratories, highlighting the necessity for a laboratory to participate in interlaboratory comparisons.¹⁹ Selecting an EQA provider who is accredited to ISO/IEC 17043:2010 is ideal as it guarantees high quality programs that are fit for purpose and assists laboratories in achieving their own accreditation.²⁰

During the SARS-CoV-2 pandemic, it is vital that laboratories participate in an EQA scheme that adheres to the ISO/IEC Guide and ISO/IEC 17043:2010 to ensure optimum performance of analytical laboratories, including instrumentation, and the competence of the analytical personnel.

While diagnostics has always played an essential role, we are now in an era where diagnostics is vitally important to aid in reducing the mortality rate from SARS-CoV-2. As such, accurate and reliable controls and reagents are even more vital to clinical lab results. In addition, superior assay methodologies can aid in the early identification of adverse outcomes, enabling timely and effective treatment plan implementation. In doing so, quality control, both internally and externally, are vital to ensure optimum performance of assays, instrumentation, laboratories, and personnel.

References

1. WebMD. Complications coronavirus can cause. https://www.webmd.com/lung/coronavirus-complications#1. Accessed July 22, 2020.
2. George A. Cytokine storm: an overreaction of the body’s immune system. https://www.newscientist.com/term/cytokine-storm/. Accessed July 22, 2020.
3. Sinha P, Matthey MA, Calfee CS. Is a “cytokine storm” relevant to COVID-19? JAMA Intern Med June 2020: 1-3. https://jamanetwork.com/journals/jamainternalmedicine/fullarticle/2767939. Accessed July 22, 2020.
4. Qin C, Zhou L, Hu Z, Zhang S, Yang S, et al. Dysregulation of immune response in patients with coronavirus 2019 (COVID-19) in Wuhan, China. Clin Infect Dis. Jul 28;71(15):762-768. https://academic.oup.com/cid/article/doi/10.1093/cid/ciaa248/5803306. Accessed July 24, 2020.
5. Del Valle DM, Kim-Schulze S, Huang HH, Beckmann M, Nirenberg S, et al. An inflammatory cytokine signature helps predict COVID-19 severity and death. The Preprint Server for Health Sciences 2020. https://www.medrxiv.org/content/10.1101/2020.05.28.20115758v1.full.pdf. Accessed July 24, 2020.
6. Tighe PJ, Ryder RR, Todd I, Faircloght LC. ELISA in the multiplex era: potentials and pitfalls. Proteomics Clin Appl 2015; 9(3-4): 406-422.
7. Schmidt RL, Straseski JA, Raphael KL, Adams AH, Lehman CM. A risk assessment of the Jaffe vs enzymatic method for creatinine measurement in an outpatient population. PLoS One 2015; 10(11).
8. Swedko PJ, Clark HD, Paramsothy K, Akbari a. Serum creatinine is an inadequate screening test for renal failure in elderly patients. JAMA 2003; 163(3): 356-360.
9. Gounden V, Jialal I. Renal function tests. Treasure Island: StatPearls Publishing; 2020. https://www.ncbi.nlm.nih.gov/books/NBK507821/. Accessed June 15, 2020.
10. Siordia JA. Epidemiology and clinical features of COVID-19: a review of current literature. Journal of Clin Virol. 2020; 127(2020): 1-7.
11. Cai Q, Huang D, Yu H, Zhu Z, Xia Z, et al. COVID-19: Abnormal liver function tests. J Hepatol. 2020; 4(6): 1-9.
12. Paliogiannis P, Zinellu A. Bilirubin levels in patients with mild and severe Covid‐19: a pooled analysis. Liver Int. 2020; 40: 1787-1788.
13. Dhungana N, Morris C, Krasowski MD. Operational impact of using a vanadate oxidase method for direct bilirubin measurements at an academic medical center clinical laboratory. Practical Laboratory Medicine 2017; 8(2017): 77-85.
14. Moriarty PM, Gorby LK, Stroes ES, Kastelein JP, Davidson M, et al. Lipoprotein(a) and its potential association with thrombosis and inflammation in COVID-19: a testable hypothesis. Current Atheroscler Rep. 2020; 22(48): 1-8.
15. Cegla J, Dermot R, Neely G, France M, Ferns G, et al. HEART UK consensus statement on lipoprotein(a): a call to action. Atherosclerosis 2019; 291: 62-70.
16. Pai S, Frater JL. Quality management and accreditation in laboratory hematology: perspectives from India. International Journal of Laboratory Hematology 2019; 41(1): 177-183.
17. Eden PR. Quality control in clinical laboratory samples. Medical Laboratory Observer. January 2015 https://www.mlo-online.com/home/article/13007888/quality-control-in-clinical-laboratory-samples. Accessed July 23, 2020.
18. International Organization for Standardization (ISO). Medical laboratories - requirements for quality and competence (ISO 15189:2012). https://www.erndim.org/store/docs/extractfromISO151892012-FESUUMUJ926264-7-5-2013.pdf. Accessed July 23, 2020.
19. World Health Organization (WHO) 10. Assessment - external quality assessment. https://extranet.who.int/lqsi/sites/default/files/attachedfiles/LQMS%2010.%20Assessment%20-%20EQA.pdf. Accessed 23 July 2020.
20. International Organization for Standardization (ISO). ISO/IEC 17043:2010 Conformity assessment - General requirements for proficiency testing. https://www.iso.org/standard/29366.html. Accessed July 23, 2020.