Review Article2022-01-01
Clinical Chemistry

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.

Ann Lab Med 2022; 42(1): 3-23

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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.

Review Article2021-11-01
Clinical Chemistry

Second-Line Tests in the Diagnosis of Adrenocorticotropic Hormone-Dependent Hypercortisolism

Silvia Pinelli , M.D., Mattia Barbot , M.D., Ph.D., Carla Scaroni , M.D., and Filippo Ceccato , M.D., Ph.D.

Ann Lab Med 2021; 41(6): 521-531

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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.

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 , 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.

Ann Lab Med 2022; 42(1): 24-35

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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.

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 , B.S.

Ann Lab Med 2022; 42(2): 121-140

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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.

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 , 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.

Ann Lab Med 2021; 41(4): 357-365

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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.

Brief Communication2021-01-01
Clinical Chemistry

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Abstract : The Barricor tube (Becton Dickinson [BD], Sunnyvale, CA, USA) was recently developed to mechanically separate plasma by increasing the centrifugation rate. We compared the Barricor tube with existing serum- and plasma-based tubes based on 35 biochemical analytes and preanalytical turnaround time (TAT). Blood samples were collected from 30 healthy volunteers in a Barricor tube, serum separating tube (SST, Vacutainer SST II Tube 8.5 mL, #368972; BD), or plasma separating tube (PST, Vacutainer PST Tube 8.0 mL, #367964; BD) in random order. Next, 27 chemistry analytes, six immunochemistry analytes, and two cardiac markers were compared using Passing-Bablok regression and the Bland-Altman method. Preanalytical TAT was measured for each tube. The Barricor tube exhibited bias exceeding the desirable limit for nine and four analytes compared with the SST and PST, respectively. The Barricor tube lactate dehydrogenase value showed a bias of -10.29% and -9.86% compared with that of the SST and PST, respectively. The preanalytical TAT of Barricor tube was 8.8 minutes, which was the shortest among the three tubes. The clinical performance of the Barricor tube was equivalent to that of the SST and PST for most analytes, with an apparent advantage in preanalytical TAT. When using the Barricor tube, the reference range needs to be changed for some analytes that exceed the desirable bias limit.

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 , B.S.

Ann Lab Med 2022; 42(5): 531-557

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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.

Original Article2022-07-01
Clinical Chemistry

Prognostic Utility of Procalcitonin, Presepsin, and the VACO Index for Predicting 30-day Mortality in Hospitalized COVID-19 Patients

Mikyoung Park , M.D., Ph.D, Mina Hur , M.D., Ph.D, Hanah Kim , M.D., Ph.D, Chae Hoon Lee , M.D., Ph.D, Jong Ho Lee , M.D., Hyung Woo Kim , M.D., and Minjeong Nam , M.D., Ph.D.

Ann Lab Med 2022; 42(4): 406-414

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Abstract : Background: Biomarkers and clinical indices have been investigated for predicting mortality in patients with coronavirus disease (COVID-19). We explored the prognostic utility of procalcitonin (PCT), presepsin, and the Veterans Health Administration COVID-19 (VACO) index for predicting 30-day-mortality in COVID-19 patients. Methods: In total, 54 hospitalized COVID-19 patients were enrolled. PCT and presepsin levels were measured using the Elecsys BRAHMS PCT assay (Roche Diagnostics GmbH, Mannheim, Germany) and HISCL Presepsin assay (Sysmex, Kobe, Japan), respectively. The VACO index was calculated based on age, sex, and comorbidities. PCT and presepsin levels and the VACO index were compared using ROC curve, Kaplan–Meier method, and reclassification analysis for the 30-day mortality. Results: ROC curve analysis was used to measure PCT and presepsin levels and the VACO index to predict 30-day mortality; the optimal cut-off values were 0.138 ng/mL for PCT, 717 pg/mL for presepsin, and 12.1% for the VACO index. On Kaplan–Meier survival analysis, hazard ratios (95% confidence interval) were 15.9 (4.1-61.3) for PCT, 26.3 (6.4-108.0) for presepsin, and 6.0 (1.7-21.1) for the VACO index. On reclassification analysis, PCT and presepsin in addition to the VACO index significantly improved the prognostic value of the index. Conclusions: This study demonstrated the prognostic utility of measuring PCT and presepsin levels and the VACO index in COVID-19 patients. The biomarkers in addition to the clinical index were more useful than the index alone for predicting clinical outcomes in COVID-19 patients.

Brief Communication2021-01-01
Clinical Chemistry

A Sensitive and Specific Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Quantification of Salivary Melatonin and Cortisol: Development and Comparison With Immunoassays

Sunghwan Shin , M.D., Hyeonju Oh , M.S., Hea Ree Park , M.D., Eun Yeon Joo , M.D., Ph.D., and Soo-Youn Lee , M.D., Ph.D.

Ann Lab Med 2021; 41(1): 108-113

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Abstract : Melatonin and cortisol are clinically important for diagnosing sleep and mood disorders. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of salivary melatonin and cortisol concentrations according to the Clinical and Laboratory Standards Institute guidelines. Additionally, we compared the LC-MS/MS assay with immunoassays, ELISA (Direct Salivary Melatonin Elisa EK-DSM, Bühlmann Laboratories AG, Schönenbuch, Switzerland) and electrochemiluminescence immunoassay (Cortisol II, Roche, Mannheim, Germany), using 121 saliva samples. The LC-MS/MS assay exhibited good performance in terms of linearity, precision, accuracy, limit of detection, lower limit of quantification, extraction recovery, carry-over, and matrix effect. The LC-MS/MS assay and immunoassays showed strong correlation (Pearson’s r=0.910 for melatonin, r=0.955 for cortisol), but demonstrated a significant mean bias of 23.2% (range 54.0–143.7%) for melatonin and 48.9% (range 59.7–184.7%) for cortisol. Our LC-MS/MS assay provided more sensitive and reliable salivary melatonin and cortisol quantification results compared with immunoassays.

Original Article2022-03-01
Clinical Chemistry

Impact of Individualized Hemolysis Management Based on Biological Variation Cut-offs in a Clinical Laboratory

Fernando Marques-Garcia , Ph.D., David Hansoe Heredero Jung , M.D., and Sandra Elena Pérez, B.D.

Ann Lab Med 2022; 42(2): 169-177

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Abstract : Background: Hemolysis is the most common type of preanalytical interference. Cut-offs based on the hemolysis index level can be established using different approaches. The Working Group for Preanalytical Phase of the European Federation of Laboratory Medicine has developed a protocol for hemolysis management based on cut-offs estimated from biological variation (BV) and the use of interpretative comments. We developed and assessed the implementation of the protocol in our laboratory. Methods: Hemolysates from whole blood were prepared following the Meites method, and pooled serum samples with known Hb concentrations were prepared. For each analyte (42 ), interferograms were generated and used to establish cut-offs: desirable analytical quality specification and reference change value. This protocol was assessed, both pre- and post-implementation, according to expert rules in the Laboratory Information System. Results: Among the analytes evaluated, we selected those that showed the highest degree of hemolysis interference: lactate dehydrogenase (LDH), aspartate aminotransferase, direct bilirubin, potassium, and folic acid. The cut-offs for LDH and direct bilirubin were the lowest. Only 28.16% of all LDH values were adequately reported in the pre-implantation retrospective study, but this percentage improved in the post-implementation stage. Conclusions: The development and implementation of a harmonized protocol for hemolysis management based on BV cut-offs and result reporting significantly improve hemolysis detection and lead to a decrease in the number of hemolyzed samples over time.