Clinical Impact of New Reference Intervals for the Roche Prolactin II Immunoassay.

Context: The Roche prolactin immunoassay is used throughout the world. It reports higher values than the Siemens immunoassay but the manufacturer-defined reference intervals are similar. Patient results are often above the Roche upper limit but within the Siemens interval, causing diagnostic confusion. Objective: Establish new reference intervals for the Roche and Siemens prolactin immunoassays. Methods: We established new reference intervals for the Roche and Siemens immunoassays using 374 specimens from healthy outpatients. We performed chart review for unnecessary testing and treatment for 298 patients in a 6-month period with at least 1 Roche prolactin value above the manufacturer-defined upper limit and below our new upper limit. Results: The new upper limit for the Roche assay was 37.8 ng/mL (females) and 22.8 ng/mL (males). The manufacturer-defined limits were 23.3 ng/mL and 15.2 ng/mL, respectively. New intervals for the Siemens assay matched the manufacturer. No cases of clinically significant pathophysiologic prolactin excess were identified in patients with values between the manufacturer-defined upper reference limit and our new Roche upper limit. Unnecessary further evaluation in these patients included 459 repeat prolactin measurements, 57 macroprolactin measurements, 39 magnetic resonance imaging studies, and 28 endocrine referrals. Eleven patients received dopamine agonists. The minimum cost of excess care using Medicare reimbursement rates was $34 134, with substantially higher amounts billed to patients and their insurance providers. Conclusion: Adoption of new upper reference limits for the Roche prolactin assay of 37.8 ng/mL (females) and 22.8 ng/mL (males) would not delay diagnosis or necessary intervention in patients with clinically significant pituitary tumors but would reduce unnecessary evaluation in patients without pathophysiologic prolactin excess.

Mistaken Identity: The Role of Autoantibodies in Endocrine Disease.

BACKGROUND: Autoimmune endocrine diseases can be thought of as a case of mistaken identity. The immune system mistakenly attacks one's own cells, as if they were foreign, which typically results in endocrine gland hypofunction and inadequate hormone production. Type 1 diabetes mellitus and autoimmune thyroid disorders (Hashimoto and Graves diseases) are the most common autoimmune endocrine disorders, while conditions such as Addison disease are encountered less frequently. Autoantibody production can precede clinical presentation, and their measurement may aid verification of an autoimmune process and guide appropriate treatment modalities. CONTENT: In this review, we discuss type 1 diabetes mellitus, autoimmune thyroid disorders, and Addison disease, emphasizing their associated autoantibodies and methods for clinical detection. We will also discuss efforts to standardize measurement of autoantibodies. CONCLUSIONS: Autoimmune endocrine disease progression may take months to years and detection of associated autoantibodies may precede clinical onset of disease. Although detection of autoantibodies is not necessary for diagnosis, they may be useful to verify an autoimmune process.

National Kidney Foundation Laboratory Engagement Working Group Recommendations for Implementing the CKD-EPI 2021 Race-Free Equations for Estimated Glomerular Filtration Rate: Practical Guidance for Clinical Laboratories.

Recognizing that race is a social and not a biological construct, healthcare professionals and the public have called for removal of race in clinical algorithms. In response, the National Kidney Foundation and the American Society of Nephrology created the Task Force on Reassessing the Inclusion of Race in Diagnosing Kidney Diseases to examine the issue and provide recommendations. The final report from the Task Force recommends calculating estimated glomerular filtration rate (eGFR) without a race coefficient using the recently published CKD-EPI 2021 creatinine (cr) and creatinine-cystatin C (cr-cys) equations. The Task Force recommends immediately replacing older eGFRcr equations (MDRD Study and CKD-EPI 2009) with the new CKD-EPI 2021 equation. In a 2019 survey by the College of American Pathologists, 23% of 6200 laboratories reporting eGFRcr used an incorrect equation that is not suitable for use with standardized creatinine measurements, 34% used the CKD-EPI 2009 equation and 43% used the MDRD Study 2006 equation re-expressed for standardized creatinine measurement. Rapid transition to using the CKD-EPI 2021 equation is an opportunity for laboratories to standardize to a single equation to eliminate differences in eGFRcr due to different equations used by different laboratories, and to report eGFR without use of race. We provide guidance to laboratories for implementing the CKD-EPI 2021 equations for both eGFRcr and eGFRcr-cys.

AACC Guidance Document on Laboratory Testing for the Assessment of Preterm Delivery.

Identifying women with preterm labor who will go on to deliver prematurely is crucial to improving outcomes for mother and baby and for saving healthcare resources. Even among those with symptoms, the number of women who deliver preterm is low, and thus the low positive predictive value (PPV) and high negative predictive value (NPV) associated with available biomarkers does not substantially reduce the uncertainty of the clinical diagnosis. While there is some promise in the use of fetal fibronectin (fFN), interleukin 6 (IL-6), or placental alpha microglobulin 1 (PAMG-1) for predicting preterm birth (PTB), their use is unlikely to provide considerable clinical value in populations with a low prevalence. To provide real clinical benefit, a biomarker must demonstrate a high PPV to allow identification of the minority of symptomatic women who will deliver prematurely. As none of the currently available biomarkers exhibit this performance characteristic, we do not recommend their routine clinical use in populations with a pre-test probability of PTB of <5%. Limiting biomarker testing to only high-risk women identified on the basis of cervical length or other characteristics will increase the pre-testprobability in the tested population, thereby improving PPV. PAMG-1 is associated with a higher PPV than fFN and may show clinical utility in populations with a higher pre-test probability, but further work is required to conclusively demonstrate improved outcomes in this patient group.

Validation study of the Access antimüllerian hormone assay for the prediction of poor ovarian response to controlled ovarian stimulation.

OBJECTIVE: To evaluate the diagnostic performance of the antimüllerian hormone (AMH) level determined using the Access AMH assay for predicting poor ovarian response (POR) defined as ≤4 oocytes retrieved, including the validation of the predefined AMH cutoff of 0.93 ng/mL in both serum and plasma. DESIGN: Prospective cohort study. SETTING: Fifteen private and academic fertility centers (14 in the United States and 1 in Canada). PATIENT(S): Women aged 21-45 years planning controlled ovarian stimulation for in vitro fertilization. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Number of oocytes retrieved, categorized as POR and normal-to-high ovarian response (non-POR). The correlation of AMH level and antral follicle count. RESULT(S): Data were available for 472 participants who completed the study (74 with POR and 398 non-POR). The mean AMH serum level among those with POR was 0.99 ng/mL (median 0.76 ng/mL) compared with 2.83 ng/mL (median 2.36 ng/mL) among the normal-to-high responders. For confirmation of the 0.93 ng/mL AMH level cutoff as a predictor of POR, a receiver operating characteristic analysis gave an area under the curve of 0.852, with corresponding sensitivity and specificity of 63.5% and 89.2%, respectively. The associated positive predictive value was 52.2% and the negative predictive value was 92.9%. The AMH plasma values demonstrated a strong correlation with AMH serum values with an r value = 0.9980. The previously established AMH cutoff of 1.77 ng/mL for antral follicle count >15 resulted in a sensitivity of 83.8% (95% confidence interval [CI] 77.7-88.5) and a specificity of 59.9% (95% CI 54.2-65.4). CONCLUSION(S): This study validated the previously established AMH cut-point for the prediction of POR. Because this cut-point may vary depending on the assay used, the specific AMH assay should be reported in the literature whenever possible.

Evaluation of 3 SARS-CoV-2 IgG Antibody Assays and Correlation with Neutralizing Antibodies.

BACKGROUND: As serologic assays for SARS-CoV-2 become more widely utilized, it is important to understand their performance characteristics and correlation with neutralizing antibodies. We evaluated 3 commonly used SARS-CoV-2 IgG assays (Abbott, DiaSorin, and EUROIMMUN) for clinical sensitivity, specificity, and correlation with neutralizing antibodies, and then compared antibody kinetics during the acute phase of infection. METHODS: Three panels of samples were tested on every assay. Sensitivity was assessed using a panel of 35 specimens serially collected from 7 patients with RT-PCR-confirmed COVID-19. Specificity was determined using 100 sera samples collected in 2018 from healthy individuals prior to the outbreak. Analytical specificity was determined using a panel of 37 samples from individuals with respiratory illnesses other than COVID-19. RESULTS: Clinical sensitivity was 91.43% (95% CI 76.94-98.20%) for Abbott, and 88.57% (95% CI 73.26-96.80%) for both DiaSorin and EUROIMMUN. Clinical specificity was 99.00% (95% CI 94.55-99.97%) for Abbott and DiaSorin and 94.00% (95% CI 87.40-97.77%) for EUROIMMUN. The IgG assays demonstrated good qualitative agreement (minimum of 94%) and good correlation between the quantitative result for each combination of assays (r2 ≥ 0.90). The neutralizing antibody response did not necessarily follow the same temporal kinetics as the IgG response and did not necessarily correlate with IgG values. CONCLUSION: The 3 IgG antibody assays demonstrated comparable performance characteristics. Importantly, a qualitative positive IgG result obtained with any of the assays was associated with the presence of neutralizing antibodies; however, neutralizing antibody concentrations did not correlate well with signal to cutoff ratios.

Myocardial Infarction Can Be Safely Excluded by High-sensitivity Troponin I Testing 3 Hours After Emergency Department Presentation.

BACKGROUND: The accuracy and speed by which acute myocardial infarction (AMI) is excluded are an important determinant of emergency department (ED) length of stay and resource utilization. While high-sensitivity troponin I (hsTnI) >99th percentile (upper reference level [URL]) represents a "rule-in" cutpoint, our purpose was to evaluate the ability of the Beckman Coulter hsTnI assay, using various level-of-quantification (LoQ) cutpoints, to rule out AMI within 3 hours of ED presentation in suspected acute coronary syndrome (ACS) patients. METHODS: This multicenter evaluation enrolled adults with >5 minutes of ACS symptoms and an electrocardiogram obtained per standard care. Exclusions were ST-segment elevation or chronic hemodialysis. After informed consent was obtained, blood samples were collected in heparin at ED admission (baseline), ≥1 to 3, ≥3 to 6, and ≥6 to 9 hours postadmission. Samples were processed and stored at -20°C within 1 hour and were tested at three independent clinical laboratories on an immunoassay system (DxI 800, Beckman Coulter). Analytic cutpoints were the URL of 17.9 ng/L and two LoQ cutpoints, defined as the 10 and 20% coefficient of variation (5.6 and 2.3 ng/L, respectively). A criterion standard MI diagnosis was adjudicated by an independent endpoint committee, blinded to hsTnI, and using the universal definition of MI. RESULTS: Of 1,049 patients meeting the entry criteria, and with baseline and 1- to 3-hour hsTnI results, 117 (11.2%) had an adjudicated final diagnosis of AMI. AMI patients were typically older, with more cardiovascular risk factors. Median (IQR) presentation time was 4 (1.6-16.0) hours after symptom onset, although AMI patients presented ~0.5 hour earlier than non-AMI. Enrollment and first blood draw occurred at a mean of ~1 hour after arrival. To evaluate the assay's rule-out performance, patients with any hsTnI > URL were considered high risk and were excluded. The remaining population (n = 829) was divided into four LoQ relative categories: both hsTnI < LoQ (Lo-Lo cohort); first hsTnI < LoQ and 2nd > LoQ (Lo-Hi cohort); first > LoQ and second < LoQ (Hi-Lo cohort); or both > LoQ (Hi-Hi cohort). In patients with any hsTnI result <20% CV LoQ (Groups 1-3), n = 231 (23.9% ruled out), AMI negative predictive value (NPV) was 100% (95% confidence interval [CI] = 98.9% to 100%). In patients with any hsTnI below the 10% LoQ, n = 611 (58% rule out), AMI NPV was 100% (95% CI = 99.5% to 100%). Of the Hi-Hi cohort (i.e., no hsTnI below the 10% LoQ, but both < URL), there were four AMI patients, NPV was 98.2% (95% CI = 95.4% to 99.3%), and sensitivity was 96.6. CONCLUSIONS: Patients presenting >3 hours after the onset of suspected ACS symptoms, with at least two Beckman Coulter Access hsTnI < URL and at least one of which is below either the 10 or the 20% LoQ, had a 100% NPV for AMI. Two hsTnI values 1 to 3 hours apart with both < URL, but also >LoQ had inadequate sensitivity and NPV.

An Intact ACTH LC-MS/MS Assay as an Arbiter of Clinically Discordant Immunoassay Results.

BACKGROUND: Measurement of plasma adrenocorticotropic hormone (ACTH) is key in the differential diagnosis of hypothalamic-pituitary-adrenal disorders. Two-site sandwich immunoassays dominate clinical testing of ACTH in North America; however, discordant results between manufacturers have been repeatedly reported. To resolve the discrepancy, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the intended measurand, biologically active intact ACTH (iACTH). METHODS: The multiple reaction monitoring LC-MS/MS assay was designed to selectively measure full-length iACTH, as well as ACTH analogs and fragments (i.e., ACTH1-24 and ACTH18-39). Epitope assignment of the Roche Elecsys antibodies was performed by MALDI-TOF mass spectrometry. A method comparison between Roche Elecsys and Siemens Immulite ACTH immunoassays was performed and clinically concordant/discordant results identified. In a subset of these samples, the iACTH concentration was determined using the LC-MS/MS method. RESULTS: The lower limit of the measuring interval of the iACTH LC-MS/MS assay was 9 pg/mL (2 pmol/L). The assay was linear from 9 to 1938 pg/mL (2 to 427 pmol/L). Epitope mapping revealed that the Roche capture and detection antibodies bound residues 9-12 and 36-39 of ACTH, respectively. The iACTH LC-MS/MS analysis demonstrated that for discordant results between 2 immunoassays studied, only the Roche results were highly positively correlated with the iACTH concentration. CONCLUSIONS: Immunoprecipitation of biologically active ACTH molecules followed by LC-MS/MS analysis enabled selective detection of iACTH and relevant biologically active fragments in plasma. Applied to the investigation of clinically discrepant results, this method can act as an arbiter of the concentration of iACTH present.

Best practices in mitigating the risk of biotin interference with laboratory testing.

Dietary biotin intake does not typically result in blood biotin concentrations that exceed interference thresholds for in vitro diagnostic tests. However, recent trends of high-dose biotin supplements and clinical trials of very high biotin doses for patients with multiple sclerosis have increased concerns about biotin interference with immunoassays. Estimates of the prevalence of high biotin intake vary, and patients may be unaware that they are taking biotin. Since 2016, 92 cases of suspected biotin interference have been reported to the US Food and Drug Administration. Immunoassays at greatest risk from biotin interference include thyroid and reproductive hormones, cardiac, and immunosuppressive drug tests. Several case studies have highlighted the challenge of biotin interference with thyroid hormone assays and the potential misdiagnosis of Graves' disease. Biotin interference should be suspected when immunoassay test results are inconsistent with clinical information; a clinically relevant biotin interference happens when the blood biotin concentration is high and the assay is sensitive to biotin. We propose a best practice workflow for laboratory scientists to evaluate discrepant immunoassay results, comprising: (1) serial dilution; (2) retesting after biotin clearance and/or repeat testing on an alternate platform; and (3) confirmation of the presence of biotin using depletion protocols or direct measurement of biotin concentrations. Efforts to increase awareness and avoid patient misdiagnosis should focus on improving guidance from manufacturers and educating patients, healthcare professionals, and laboratory staff. Best practice guidance for laboratory staff and healthcare professionals would also provide much-needed information on the prevention, detection, and management of biotin interference.

Peritoneal and Pleural Fluid Chemistry Measurements Performed on Three Chemistry Platforms.

BACKGROUND: Chemistry testing is requested for body fluid (BF) specimens despite the lack of assays approved by the US Food and Drug Administration (FDA). The criteria for categorizing fluids as transudate or exudate are not validated across analyzers. OBJECTIVE: To compare BF chemical analysis and classification by different analyzers. METHODS: We analyzed 10 pleural and 18 peritoneal fluids with corresponding plasma specimens using the Vitros 5,1 FS; Abbott ARCHITECT ci8200; and Roche Modular P platforms. Total protein (TP) and lactate dehydrogenase (LDH) were measured for pleural fluids. Light's criteria were applied. Albumin was measured for peritoneal specimens, and the plasma-ascites-albumin gradient was calculated. RESULTS: TP results showed agreement. The Vitros LDH assay produced higher fluid:plasma ratios. Classification by Light's criteria resulted in 1 discrepancy (ARCHITECT). Albumin results showed agreement. There were 2 discrepant gradient interpretations (Vitros). CONCLUSIONS: These data suggest that analyses of pleural and peritoneal fluids using these platforms are diagnostically interchangeable.

Reference Intervals: Comparison of Calculation Methods and Evaluation of Procedures for Merging Reference Measurements From Two US Medical Centers.

OBJECTIVES: To analyze consistency of reference limits and widths of reference intervals (RIs) calculated by six procedures and evaluate a protocol for merging intrainstitutional reference data. METHODS: The differences between reference limits were compared with "optimal" bias goals. Also, widths of the RIs were compared. RIs were calculated using Mayo-SAS quantile, EP Evaluator, and four International Federation of Clinical Chemistry and Laboratory Medicine methods: parametric and nonparametric (NP) with and without latent abnormal values exclusion (LAVE). Regression parameters from cotested samples were evaluated for harmonizing intrainstitutional reference data. RESULTS: Mayo-SAS quintile, LAVE(-)NP, and EP Evaluator generated similar RIs, but these RIs often were wider than RIs from parametric procedures. LAVE procedures generated narrower RIs for nutritional and inflammatory markers. Transformation with regression parameters did not ensure homogeneity of merged data. CONCLUSIONS: Parametric methods are recommended when inappropriate values cannot be excluded. The nonparametric procedures may generate wider RIs. Data sets larger than 200 are recommended for robust estimates. Caution should be exercised when merging intrainstitutional data.

Multicenter evaluation of the Access AMH antimüllerian hormone assay for the prediction of antral follicle count and poor ovarian response to controlled ovarian stimulation.

OBJECTIVE: To evaluate a new fully automated antimüllerian hormone (AMH) assay for prediction of poor ovarian response (POR) to ovarian stimulation defined as four or fewer oocytes retrieved. DESIGN: Prospective cohort study. SETTING: Thirteen private and academic fertility centers in the United States. PATIENTS(S): A total of 178 women undergoing their first in vitro fertilization (IVF) cycle eligible for the study were consented and enrolled, with data available from 160 women for prediction of POR and 164 women for AMH correlation with antral follicle count (AFC). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Cutoff point for AMH that predicts POR. Correlation of AMH with AFC, and cutoff point for AMH that correlates with antral follicle count >15. RESULT(S): The mean AMH among the poor responders was 0.74 ng/mL, compared with 3.20 ng/mL for normal to high responders. The AMH cutoff at 90% specificity for predicting POR with the use of the receiver operating characteristic (ROC) curve was 0.93 ng/mL, with an associated sensitivity of 74.1%. For prediction of POR, ROC analysis showed that AMH (area under the ROC curve [AUC] = 0.929) was significantly better than FSH (AUC = 0.615; P<.0001). AMH was positively correlated with AFC (Spearman rho = 0.756). The AMH at 90% sensitivity for AFC >15 was 1.75, with specificity of 59.1%. CONCLUSION(S): A fully automated AMH assay can be a useful biomarker for predicting POR in IVF cycles. Because AMH cutoff points vary depending on the assay used, future studies should continue to calibrate test results to clinically important outcomes.

Does Reverse Triiodothyronine Testing Have Clinical Utility? An Analysis of Practice Variation Based on Order Data from a National Reference Laboratory.

BACKGROUND: Clinical laboratories are under pressure to increase value by improving test utilization. The clinical utility of reverse triiodothyronine (rT3) is controversial. A study was conducted to identify order patterns that might suggest inappropriate utilization of rT3. METHODS: All orders for thyroid tests placed over a period of one year at a national reference laboratory were reviewed. Order patterns by client (hospital) and by provider were analyzed. A Pareto analysis was conducted to determine the percentage of orders placed as a function of the percentage of providers. A systematic review of the indexed literature and an informal review of the web were conducted to identify indications for rT3 testing. RESULTS: There were 402,386 orders for 447,664 thyroid tests, including 91,767 orders for rT3. These orders were placed by 60,733 providers located at 1139 different organizations. Only 20% of providers who ordered thyroid tests placed an order for rT3. Of those who placed an order for rT3, 95% placed two orders or fewer for rT3. One hundred providers (0.1% of the 60,733 providers who placed orders for thyroid tests) accounted for 29.5% of the orders for rT3. Of the 100 providers, 60 with the highest order volumes for rT3 were classified as practitioners of functional medicine. A systematic review of Medline found little evidence to support the high volumes of orders for rT3. A survey of Web sites for functional medicine suggests that rT3 is useful for the diagnosis of rT3 dominance and can be used to direct triiodothyronine replacement therapy. CONCLUSIONS: There is wide practice variation in rT3 testing. A high proportion of tests are ordered by a relatively small proportion of providers. There is little evidence to support high volumes of rT3 testing placed by some practitioners.

Measuring estrogens in women, men, and children: Recent advances 2012-2017.

The measurement of estrogens is important for diagnosing and monitoring the health of women, men, and children. For example, for postmenopausal women or women undergoing treatment for breast cancer with aromatase inhibitors, the measurement of extremely low concentrations of estrogens in serum, especially estradiol, is problematic but essential for proper medical care. Achieving superb analytical sensitivity and specificity has been and continues to be a challenge for the clinical laboratory, but is a challenge that is being taken seriously. Focusing on publications from 2012 to 2017, this review will provide an overview of recent research in the development of methods to accurately and precisely measure estrogens, including a variety of estrogen metabolites. Additionally, the latest in clinical research involving estrogen measurement in women, men, and children will be presented to provide an update on the association of estrogens with diseases or conditions such as breast cancer, precocious puberty, infertility, and pregnancy. This research update will provide context as to why estrogen measurement is important and why laboratories are working hard to support the recommendations made by the Endocrine Society regarding estrogen measurement.

How low can you go? Analytical performance of five automated testosterone immunoassays.

BACKGROUND: Testosterone is commonly measured using immunoassays, yet concerns with the accuracy and quality of testing by these methods exist, particularly for low testosterone concentrations. Study objectives were to evaluate selective performance characteristics, including functional sensitivity (FS), of 5 automated immunoassays for total testosterone. METHODS: FS, imprecision, assay interference, limit of blank, linearity, and accuracy were assessed using the Abbott ARCHITECT i2000SR, SIEMENS ADVIA Centaur and IMMULITE 2000, Beckman Coulter DxI 800, and Roche MODULAR E170. Comparisons to an in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were performed using patient samples from men, women, boys, and girls. RESULTS: FS at 20% coefficient of variation (CV) for the ARCHITECT, Centaur, DxI, E170 and IMMULITE assays were 0.14, 1.23, 0.36, 0.77, 3.49 nmol/L, respectively. Total CVs for the 5-day imprecision study were ≤ 9.0% for all methods. All assays met manufacturer's claims for hemolysis, icterus, and lipemia interference and limit of blank. Dilution linearity studies had deviations from the target recoveries ranging from 3.4% (ARCHITECT) to 14.3% (DxI). Using National Institute of Standards and Technology Standard Reference Material 971, recoveries ranged from 79.2-149.2% (DxI, male and female, respectively). When compared to LC-MS/MS, more immunoassays under-recovered in men and women and over-recovered in boys and girls. Slopes ranged from 0.71 (IMMULITE, women) to 1.35 (DxI, boys). The combined average for percent bias was higher in boys (28.0%) than men (11.6%), women (22.8%), and girls (25.7%). CONCLUSIONS: Challenges with accurately measuring testosterone appear to remain for some immunoassays, but not all. While most immunoassays remain optimized for concentrations observed in healthy men, some showed acceptable performance when challenged at lower concentrations.