Hormone Immunoassay Interference: A 2021 Update
Khaldoun Ghazal 
, Pharm., Ph.D., Severine Brabant , M.D., Dominique Prie , M.D., Ph.D., and Marie-Liesse Piketty , Pharm., Ph.D.
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Review Article2022-01-01 Clinical Chemistry
Abstract : Immunoassays are powerful qualitative and quantitative analytical techniques. Since the first description of an immunoassay method in 1959, advances have been made in assay designs and analytical characteristics, opening the door for their widespread implementation in clinical laboratories. Clinical endocrinology is closely linked to laboratory medicine because hormone quantification is important for the diagnosis, treatment, and prognosis of endocrine disorders. Several interferences in immunoassays have been identified through the years; although some are no longer encountered in daily practice, cross-reaction, heterophile antibodies, biotin, and anti-analyte antibodies still cause problems. Newer interferences are also emerging with the development of new therapies. The interfering substance may be exogenous (e.g., a drug or substance absorbed by the patient) or endogenous (e.g., antibodies produced by the patient), and the bias caused by interference can be positive or negative. The consequences of interference can be deleterious when clinicians consider erroneous results to establish a diagnosis, leading to unnecessary explorations or inappropriate treatments. Clinical laboratories and manufacturers continue to investigate methods for the detection, elimination, and prevention of interferences. However, no system is completely devoid of such incidents. In this review, we focus on the analytical interferences encountered in daily practice and possible solutions for their detection or elimination.
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Original Article2022-03-01 Clinical Chemistry
Biomarker Rule-in or Rule-out in Patients With Acute Diseases for Validation of Acute Kidney Injury in the Emergency Department (BRAVA): A Multicenter Study Evaluating Urinary TIMP-2/IGFBP7
Hyun Suk Yang
Ann Lab Med 2022; 42(2): 178-187, M.D., Ph.D., Mina Hur , M.D., Ph.D., Kyeong Ryong Lee , M.D., Ph.D., Hanah Kim , M.D., Ph.D., Hahn Young Kim , M.D., Ph.D., Jong Won Kim , M.D., Ph.D., Mui Teng Chua , M.D., Win Sen Kuan , M.D., Horng Ruey Chua , M.D., Chagriya Kitiyakara , M.D., Phatthranit Phattharapornjaroen , M.D., Anchalee Chittamma , Ph.D., Thiyapha Werayachankul , M.Sc., Urmila Anandh , M.D., Sanjeeva Herath , M.B., Ch.B., Zoltan Endre , M.D., Ph.D., Andrea Rita Horvath , M.D., Ph.D., Paola Antonini , M.D., and Salvatore Di Somma , M.D., Ph.D. on behalf of the GREAT NetworkAbstract : Background: Urine tissue inhibitor of metalloproteinases-2/insulin-like growth factor-binding protein 7 (TIMP-2/IGFBP7) (NephroCheck, Ortho Clinical Diagnostics, Raritan, NJ, USA) is a US Food and Drug Administration-approved biomarker for risk assessment of acute kidney injury (AKI) in critically ill adult patients in intensive care units; however, its clinical impact in the emergency department (ED) remains unproven. We evaluated the utility of NephroCheck for predicting AKI development and short-term mortality in the ED. Methods: This was a prospective, observational, five-center international study. We consecutively enrolled ED patients admitted with ≥30% risk of AKI development (assessed by ED physician: ED score) or acute diseases. Serum creatinine was tested on ED arrival (T0), day 1, and day 2 (T48); urine for NephroCheck was collected at T0 and T48. We performed ROC curve and reclassification analyses. Results: Among the 529 patients enrolled (213 females; median age, 65 years), AKI developed in 59 (11.2%) patients. The T0 NephroCheck value was higher in the AKI group than in the non-AKI group (median 0.77 vs. 0.29 (ng/m)2/1,000, P=0.001), and better predicted AKI development than the ED score (area under the curve [AUC], 0.64 vs. 0.53; P=0.04). In reclassification analyses, adding NephroCheck to the ED score improved the prediction of AKI development (P
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Review Article2021-11-01 Clinical Chemistry
Second-Line Tests in the Diagnosis of Adrenocorticotropic Hormone-Dependent Hypercortisolism
Silvia Pinelli
Ann Lab Med 2021; 41(6): 521-531, M.D., Mattia Barbot , M.D., Ph.D., Carla Scaroni , M.D., and Filippo Ceccato , M.D., Ph.D.Abstract : Cushing’s syndrome (CS) is a rare disease caused by chronic and excessive cortisol secretion. When adrenocorticotropin hormone (ACTH) is measurable, autonomous adrenal cortisol secretion could be reasonably ruled out in a differential diagnosis of CS. ACTH-dependent CS accounts for 80%–85% of cases and involves cortisol production stimulated by uncontrolled pituitary or ectopic ACTH secretion. Pituitary adenoma is not detected in up to one-third of cases with pituitary ACTH secretion, whereas cases of CS due to ectopic ACTH secretion may be associated with either malignant neoplasia (such as small cell lung carcinoma) or less aggressive neuroendocrine tumors, exhibiting only the typical symptoms and signs of CS. Since the differential diagnosis of ACTH-dependent CS may be a challenge, many strategies have been proposed. Since none of the available tests show 100% diagnostic accuracy, a step-by-step approach combining several diagnostic tools and a multidisciplinary evaluation in a referral center is suggested. In this review, we present a clinical case to demonstrate the diagnostic work-up of ACTH-dependent CS. We describe the most commonly used dynamic tests, as well as the applications of conventional or nuclear imaging and invasive procedures.
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Review Article2022-03-01 Clinical Chemistry
Review of the Use of Liquid Chromatography-Tandem Mass Spectrometry in Clinical Laboratories: Part I-Development
Brian A. Rappold
Ann Lab Med 2022; 42(2): 121-140 , B.S.Abstract : The process of method development for a diagnostic assay based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) involves several disparate technologies and specialties. Additionally, method development details are typically not disclosed in journal publications. Method developers may need to search widely for pertinent information on their assay(s). This review summarizes the current practices and procedures in method development. Additionally, it probes aspects of method development that are generally not discussed, such as how exactly to calibrate an assay or where to place quality controls, using examples from the literature. This review intends to provide a comprehensive resource and induce critical thinking around the experiments for and execution of developing a clinically meaningful LC-MS/MS assay.
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Original Article2022-01-01 Clinical Chemistry
Comprehensive Laboratory Data Analysis to Predict the Clinical Severity of Coronavirus Disease 2019 in 1,952 Patients in Daegu, Korea
Eun-Hyung Yoo
Ann Lab Med 2022; 42(1): 24-35, M.D., Ph.D., Soon Hee Chang , M.D., Ph.D., Do-Young Song , M.D., Ph.D., Chae Hoon Lee , M.D., Ph.D., Gyu Young Cheong , M.D., Ph.D., Sunggyun Park , M.D., Ph.D., Jae Hee Lee , M.D., Ph.D., Sooin Lee , M.D., Ph.D., Sang-Gyu Kwak , Ph.D., Chang-Ho Jeon , M.D., Ph.D., and Kyung Eun Song , M.D., Ph.D.Abstract : Background: Laboratory parameter abnormalities are commonly observed in COVID-19 patients; however, their clinical significance remains controversial. We assessed the prevalence, characteristics, and clinical impact of laboratory parameters in COVID-19 patients hospitalized in Daegu, Korea. Methods: We investigated the clinical and laboratory parameters of 1,952 COVID-19 patients on admission in nine hospitals in Daegu, Korea. The average patient age was 58.1 years, and 700 (35.9%) patients were men. The patients were classified into mild (N=1,612), moderate (N=294), and severe (N=46) disease groups based on clinical severity scores. We used chi-square test, multiple comparison analysis, and multinomial logistic regression to evaluate the correlation between laboratory parameters and disease severity. Results: Laboratory parameters on admission in the three disease groups were significantly different in terms of hematologic (Hb, Hct, white blood cell count, lymphocyte%, and platelet count), coagulation (prothrombin time and activated partial thromboplastin time), biochemical (albumin, aspartate aminotransferase, alanine aminotransferase, lactate, blood urea nitrogen, creatinine, and electrolytes), inflammatory (C-reactive protein and procalcitonin), cardiac (creatinine kinase MB isoenzyme and troponin I), and molecular virologic (Ct value of SARS-CoV-2 RdRP gene) parameters. Relative lymphopenia, prothrombin time prolongation, and hypoalbuminemia were significant indicators of COVID-19 severity. Patients with both hypoalbuminemia and lymphopenia had a higher risk of severe COVID-19. Conclusions: Laboratory parameter abnormalities on admission are common, are significantly associated with clinical severity, and can serve as independent predictors of COVID-19 severity. Monitoring the laboratory parameters, including albumin and lymphocyte count, is crucial for timely treatment of COVID-19.
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Original Article2021-07-01 Clinical Chemistry
Predictive Value of Plasma NGAL:Hepcidin-25 for Major Adverse Kidney Events After Cardiac Surgery with Cardiopulmonary Bypass: A Pilot Study
Christian Albert
Ann Lab Med 2021; 41(4): 357-365, M.D., Michael Haase , M.D., Annemarie Albert , M.D., Martin Ernst , M.D., Siegfried Kropf , Ph.D., Rinaldo Bellomo , M.D., Sabine Westphal , M.D., Rüdiger C. Braun-Dullaeus, M.D., Anja Haase-Fielitz , Pharm.D., and Saban Elitok , M.D.Abstract : Background: Neutrophil gelatinase-associated lipocalin (NGAL) and hepcidin-25 are involved in catalytic iron-related kidney injury after cardiac surgery with cardiopulmonary bypass. We explored the predictive value of plasma NGAL, plasma hepcidin-25, and the plasma NGAL:hepcidin-25 ratio for major adverse kidney events (MAKE) after cardiac surgery. Methods: We compared the predictive value of plasma NGAL, hepcidin-25, and plasma NGAL:hepcidin-25 with that of serum creatinine (Cr) and urinary output and protein for primary-endpoint MAKE (acute kidney injury [AKI] stages 2 and 3, persistent AKI >48 hours, acute dialysis, and in-hospital mortality) and secondary-endpoint AKI in 100 cardiac surgery patients at intensive care unit (ICU) admission. We performed ROC curve, logistic regression, and reclassification analyses. Results: At ICU admission, plasma NGAL, plasma NGAL:hepcidin-25, plasma interleukin-6, and Cr predicted MAKE (area under the ROC curve [AUC]: 0.77, 0.79, 0.74, and 0.74, respectively) and AKI (0.73, 0.89, 0.70, and 0.69). For AKI prediction, plasma NGAL:hepcidin-25 had a higher discriminatory power than Cr (AUC difference 0.26 [95% CI 0.00–0.53]). Urinary output and protein, plasma lactate, C-reactive protein, creatine kinase myocardial band, and brain natriuretic peptide did not predict MAKE or AKI (AUC <0.70). Only plasma NGAL:hepcidin-25 correctly reclassified patients according to their MAKE and AKI status (category-free net reclassification improvement: 0.82 [95% CI 0.12–1.52], 1.03 [0.29–1.77]). After adjustment to the Cleveland risk score, plasma NGAL:hepcidin-25 ≥0.9 independently predicted MAKE (adjusted odds ratio 16.34 [95% CI 1.77–150.49], P=0.014). Conclusions: Plasma NGAL:hepcidin-25 is a promising marker for predicting postoperative MAKE.
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Original Article2021-05-01 Clinical Chemistry
Immunosuppressive Drug Measurement by Liquid Chromatography Coupled to Tandem Mass Spectrometry: Interlaboratory Comparison in the Korean Clinical Laboratories
Hyun-Ki Kim
Ann Lab Med 2021; 41(3): 268-276, M.D., Hyung-Doo Park , M.D., Sang-Guk Lee , M.D., Hyojin Chae , M.D., Sang Hoon Song , M.D., Yong-Wha Lee , M.D., Yeo-Min Yun , M.D., Sunhyun Ahn , M.D., Serim Kim , M.D., Sun Min Lee , M.D., Soo-Youn Lee , M.D., and Sail Chun , M.D.; on behalf of the Clinical Mass Spectrometry Research Committee of the Korean Society of Clinical ChemistryAbstract : Background: Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is increasingly used for immunosuppressive drug tests. However, most LC-MS/MS tests are laboratory-developed and their agreement is unknown in different Korean laboratories. This interlaboratory comparison study evaluated test reproducibility and identified potential error sources. Methods: Test samples containing three concentrations of tacrolimus, sirolimus, everolimus, cyclosporine, and mycophenolic acid were prepared by pooling surplus samples from patients undergoing routine therapeutic drug monitoring and tested in duplicate in the participating 10 clinical laboratories. Reconstitution and storage experiments were conducted for the commonly used commercial calibrator set. The robust estimators of reproducibility parameters were calculated. Spearman’s rank correlation coefficient (rho, ρ) was used to evaluate the correlation between drugs. Multiple linear regression was used to determine whether the experimental conditions alter the calibration curves. Results: The reproducibility coefficient of variation exceeded 10% only for sirolimus concentrations 1 and 2 (10.8% and 12.5%, respectively) and everolimus concentrations 1 and 2 (12.3% and 11.4%, respectively). The percent difference values showed weak correlations between sirolimus and everolimus (ρ=0.334, P =0.175). The everolimus calibration curve slope was significantly altered after reconstitution following prolonged 5°C storage (P =0.015 for 14 days; P =0.025 for 28 days); the expected differences at 6 ng/mL were 0.598% for 14 days and 0.384% for 28 days. Conclusions: LC-MS/MS test reproducibility for immunosuppressive drugs seems to be good in the Korean clinical laboratories. Continuous efforts are required to achieve test standardization and harmonization, especially for sirolimus and everolimus.
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Review Article2022-09-01 Clinical Chemistry
Review of the Use of Liquid Chromatography-Tandem Mass Spectrometry in Clinical Laboratories: Part II–Operations
Brian A. Rappold
Ann Lab Med 2022; 42(5): 531-557, B.S.Abstract : Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly utilized in clinical laboratories because it has advantages in terms of specificity and sensitivity over other analytical technologies. These advantages come with additional responsibilities and challenges given that many assays and platforms are not provided to laboratories as a single kit or device. The skills, staff, and assays used in LC-MS/MS are internally developed by the laboratory, with relatively few exceptions. Hence, a laboratory that deploys LC-MS/MS assays must be conscientious of the practices and procedures adopted to overcome the challenges associated with the technology. This review discusses the post-development landscape of LC-MS/MS assays, including validation, quality assurance, operations, and troubleshooting. The content knowledge of LC-MS/MS users is quite broad and deep and spans multiple scientific fields, including biology, clinical chemistry, chromatography, engineering, and MS. However, there are no formal academic programs or specific literature to train laboratory staff on the fundamentals of LC-MS/MS beyond the reports on method development. Therefore, depending on their experience level, some readers may be familiar with aspects of the laboratory practices described herein, while others may be not. This review endeavors to assemble aspects of LC-MS/MS operations in the clinical laboratory to provide a framework for the thoughtful development and execution of LC-MS/MS applications.
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Brief Communication2021-05-01 Clinical Chemistry
Serum Ferritin as a Diagnostic Biomarker for Kawasaki Disease
Sung Hoon Kim
Ann Lab Med 2021; 41(3): 318-322, M.D., Eun Song Song , M.D., Somy Yoon , Ph.D., Gwang Hyeon Eom , Ph.D., Gaeun Kang , M.D., and Young Kuk Cho , M.D.Abstract : Diagnosis of Kawasaki disease (KD) is occasionally delayed because it is solely based on clinical symptoms. Previous studies have attempted to identify diagnostic biomarkers for KD. Recently, patients with KD were reported to have elevated serum ferritin levels. We investigated the usefulness of the serum ferritin level as a diagnostic biomarker for distinguishing KD from other acute febrile illnesses. Blood samples were obtained from pediatric patients with KD (N=77) and those with other acute febrile illnesses (N=32) between December 2007 and June 2011 for measuring various laboratory parameters, including serum ferritin levels. In patients with KD, laboratory tests were performed at diagnosis and repeated at 2, 14, and 56 days after intravenous immunoglobulin treatment. At the time of diagnosis, serum ferritin levels in patients with KD (188.8 μg/L) were significantly higher than those in patients with other acute febrile illnesses (106.8 μg/L, P=0.003). The serum ferritin cut-off value of 120.8 μg/L effectively distinguished patients with KD from those with other acute febrile illnesses, with a sensitivity and specificity of 74.5% and 83.3%, respectively. Serum ferritin may be a useful biomarker to distinguish KD from other acute febrile illnesses.
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Original Article2021-09-01 Clinical Chemistry
Internal Quality Control Data of Urine Reagent Strip Tests and Derivation of Control Rules Based on Sigma Metrics
Haeil Park
Ann Lab Med 2021; 41(5): 447-454, M.D., Ph.D. and Younsuk Ko , M.T.Abstract : Background: Urine reagent strip test (URST) results are semi-quantitative; therefore, the precision of URSTs is evaluated as the proportion of categorical results from repeated measurements of a sample that are concordant with an expected result. However, URSTs have quantitative readout values before ordinal results challenging statistical monitoring for internal quality control (IQC) with control rules. This study aimed to determine the sigma metric of URSTs and derive appropriate control rules for IQC. Methods: The URiSCAN Super Plus fully automated urine analyzer (YD Diagnostics, Yongin, Korea) was used for URSTs. Change in reflectance rate (change %R) data from IQC for URSTs performed between November 2018 and May 2020 were analyzed. Red blood cells, bilirubin, urobilinogen, ketones, protein, glucose, leukocytes, and pH were measured from 2-3 levels of control materials. The total allowable error (TEa) for a grade was the difference in midpoints of a predefined change %R range between two adjacent grades. The sigma metric was calculated as TEa/SD. Sigma metric-based control rules were determined with Westgard EZ Rules 3 software (Westgard QC, Madison, WI, USA). Results: Seven out of the eight analytes had a sigma metric >4 in the control materials with a negative grade (-), which were closer to the cut-offs. Corresponding control rules ranged from 12.5s to 13.5s. Conclusions: Although the URST is a semi-quantitative test, statistical IQC can be performed using the readout values. According to the sigma metric, control rules recommended for URST IQC in routine clinical practice are 12.5s to 13.5s.
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