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.

Reported Awareness and Adoption of 2021 Estimated Glomerular Filtration Rate Equations Among US Clinical Laboratories, March 2022.

This study includes clinical laboratories that participated in the first general chemistry proficiency testing survey in 2022 to assess awareness and adoption of new equations from the Chronic Kidney Disease Epidemiology Collaboration for estimated glomerular filtration rate (eGFR) that eliminated race-adjustment factors, including one based on creatinine and one based on creatinine and cystatin C.

Urine phosphoethanolamine is a specific biomarker for hypophosphatasia in adults.

OBJECTIVES: We investigated the utility of urine phosphoethanolamine (PEA) as a marker to aid in diagnosing and/or confirming hypophosphatasia (HPP) in adults and for monitoring patients on enzyme replacement therapy (ERT). METHODS: Data was collected from seventy-eight adults who were referred to the Vanderbilt Program for Metabolic Bone Disease for evaluation of a possible or confirmatory HPP diagnosis between July 2014 through December 2019. Fifty-nine patients were diagnosed with HPP and nineteen were excluded from a diagnosis of HPP. The urine PEA results of those patients with a confirmed diagnosis of HPP and those patients with a diagnosis of HPP excluded were captured and compared to other laboratory and clinical parameters consistent with HPP, including alkaline phosphatase (ALP) activity, plasma pyridoxal 5'-phosphate (PLP), the presence of musculoskeletal abnormalities, and genetic testing for pathogenic mutations in ALPL. RESULTS: Initial urine PEA values in patients in our HPP cohort and not on ERT were significantly higher (median = 150.0 nmol/mg creatinine, IQR = 82.0-202.0) compared patients in our HPP negative group (median 18.0 nmol/mg creatinine, IQR = 14.0-30.0, p < 0.0001) and higher than patients on ERT (median 65.0 nmol/mg creatinine, IQR = 45.3-79.8). Patients who began ERT had a decline in urine PEA levels after treatment with a mean decrease of 68.1 %. Plasma ALP levels were significantly lower in the group of patients with HPP and not on ERT group (median = 24.0 U/L, IQR = 15.0-29.50) compared to the patients without HPP (median = 45.50 U/L, IQR = 34.0-62.0;) and plasma PLP levels were significantly higher in the HPP non-ERT group (median = 284.0 nmol/L, IQR = 141.0-469.4) compared to the patients without HPP (median = 97.5 nmol/L, IQR = 43.7-206.0;). The area under the curve (AUC) of urine PEA, ALP, and PLP to distinguish between HPP and non-HPP patients is 0.968, 0.927 and 0.781, respectively, in our cohort. Urine PEA had 100 % specificity (95 % CI of 83.2 % to 100.0 %) for diagnosing HPP at a value >53.50 nmol/mg creatinine with a sensitivity of 88.4 %; 95%CI 75.5 to 94.9 %. ALP had a 100 % specificity (95 % CI of 82.4 % to 100.0 %) for diagnosing HPP at a value <30.5 U/L with a sensitivity of 77.2 %; (95%CI 64.8 to 86.2 %). PLP had a 100 % specificity (95 % CI of 81.6 % to 100.0 %) for diagnosing HPP at a value >436 nmol/L with a sensitivity of 26.9 %; (95%CI 16.8 to 40.3 %). The most common pathogenic or likely pathogenic mutations in our cohort were c.1250A>G (p.Asn417Ser), c.1133A>T (p.Asp378Val), c.881A>C (p.Asp294Ala), c.1171C>T (p.Arg391Cys), and c.571G>A, (p.Glu191Lys). CONCLUSIONS: Urine PEA is a promising diagnostic and confirmatory marker for HPP in patients undergoing investigation for HPP. Urine PEA also has potential use as a marker to monitor ERT compliance. Future studies are necessary to evaluate the association between PEA levels and clinical outcomes.

AACC Guidance Document on the Use of Point-of-Care Testing in Fertility and Reproduction.

BACKGROUND: The AACC Academy revised the reproductive testing section of the Laboratory Medicine Practice Guidelines: Evidence-Based Practice for Point-of-Care Testing (POCT) published in 2007. METHODS: A panel of Academy members with expertise in POCT and laboratory medicine was formed to develop guidance for the use of POCT in reproductive health, specifically ovulation, pregnancy, premature rupture of membranes (PROM), and high-risk deliveries. The committee was supplemented with clinicians having Emergency Medicine and Obstetrics/Gynecology training. RESULTS: Key recommendations include the following. First, urine luteinizing hormone (LH) tests are accurate and reliable predictors of ovulation. Studies have shown that the use of ovulation predicting kits may improve the likelihood of conception among healthy fertile women seeking pregnancy. Urinary LH point-of-care testing demonstrates a comparable performance among other ovulation monitoring methods for timing intrauterine insemination and confirming sufficient ovulation induction before oocyte retrieval during in vitro fertilization. Second, pregnancy POCT should be considered in clinical situations where rapid diagnosis of pregnancy is needed for treatment decisions, and laboratory analysis cannot meet the required turnaround time. Third, PROM testing using commercial kits alone is not recommended without clinical signs of rupture of membranes, such as leakage of amniotic fluid from the cervical opening. Finally, fetal scalp lactate is used more than fetal scalp pH for fetal acidosis due to higher success rate and low volume of sample required. CONCLUSIONS: This revision of the AACC Academy POCT guidelines provides recommendations for best practice use of POCT in fertility and reproduction.

Voxelotor Treatment Interferes With Quantitative and Qualitative Hemoglobin Variant Analysis in Multiple Sickle Cell Disease Genotypes.

OBJECTIVES: Voxelotor was recently approved for use in the United States as a treatment for sickle cell disease (SCD) and has been shown to interfere with the quantitation of hemoglobin (Hb) S percentage. This study aimed to determine the effect of voxelotor on the quantitation of hemoglobin variant levels in patients with multiple SCD genotypes. METHODS: In vitro experiments were performed to assess the impact of voxelotor treatment on hemoglobin variant testing. Whole blood samples were incubated with voxelotor and then analyzed by routinely used quantitative and qualitative clinical laboratory methods (high-performance liquid chromatography [HPLC], capillary zone electrophoresis [CZE], and acid and alkaline electrophoresis). RESULTS: Voxelotor modified the α-globin chain of multiple hemoglobins, including HbA, HbS, HbC, HbD-Punjab, HbE, HbA2, and HbF. These voxelotor-hemoglobin complexes prevented accurate quantitation of multiple hemoglobin species, including HbS, by HPLC and CZE. CONCLUSIONS: Technical limitations in quantifying HbS percentage may preclude the use of HPLC or CZE for monitoring patients treated with voxelotor. Furthermore, it is unclear whether HbS-voxelotor complexes are clinically equivalent to HbS. Consensus guidelines for reporting hemoglobin variant percentages for patients taking voxelotor are needed, as these values are necessary for determining the number of RBC units to exchange in acute situations.

Is the Climb Worth the View? The Savings/Alert Ratio for Reducing Vitamin D Testing.

BACKGROUND: Despite guideline recommendations, vitamin D testing has increased substantially. Clinical decision support (CDS) presents an opportunity to reduce inappropriate laboratory testing. OBJECTIVES AND METHODS: To reduce inappropriate testing of vitamin D at the Vanderbilt University Medical Center, a CDS assigned providers to receive or not receive an electronic alert each time a 25-hydroxyvitamin D assay was ordered for an adult patient unless the order was associated with a diagnosis in the patient's chart for which vitamin D testing is recommended. The CDS ran for 80 days, collecting data on number of tests, provider information, and basic patient demographics. RESULTS: During the 80 days, providers placed 12,368 orders for 25-hydroxyvitamin D. The intervention group ordered a vitamin D assay and received the alert for potentially inappropriate testing 2,181 times and completed the 25-hydroxyvitamin D order in 89.9% of encounters, while the control group ordered a vitamin D assay (without receiving an alert) 2,032 times and completed the order in 98.1% of encounters, for an absolute reduction of testing of 8% (p < 0.001). CONCLUSION: This CDS reduced vitamin D ordering by utilizing a soft-stop approach. At a charge of $179.00 per test and a cost to the laboratory of $4.20 per test, each display of the alert led to an average reduction of $14.70 in charges and of $0.34 in spending by the laboratory (the savings/alert ratio). By describing the effectiveness of an electronic alert in terms of the savings/alert ratio, the impact of this intervention can be better appreciated and compared with other interventions.

USE OF CONTINUOUS GLUCOSE MONITORING LEADS TO DIAGNOSIS OF HEMOGLOBIN C TRAIT IN A PATIENT WITH DISCREPANT HEMOGLOBIN A1C AND SELF-MONITORED BLOOD GLUCOSE.

OBJECTIVE: Hemoglobin A1c (HbA1c) is a useful tool for the diagnosis and management of diabetes mellitus and generally an excellent marker of overall glycemic control for the preceding 8 to 12 weeks; however, the test is not without its pitfalls. A suspicion of falsely high or low HbA1c should prompt clinicians to evaluate for possible causes. Here, we present the novel use of continuous glucose monitoring (CGM) in a case of a 49-year-old African American woman with discrepant HbA1c and self-monitored blood glucose (SMBG). CGM data suggested that the HbA1c values were falsely elevated, and subsequent evaluation led to a diagnosis of hemoglobin C (HbC) trait. METHODS: Case identified is described. Point-of-care (POC) and central laboratory HbA1c were measured on the Bayer DCA 2000 and Bio-Rad Variant II Turbo ion-exchange high-performance liquid chromatography platforms, respectively. RESULTS: The patient had an elevated POC HbA1c of 10.8% (95 mmol/mol) and a central laboratory HbA1c of 9.2% (77 mmol/mol). The patient's glucose meter measured an average glucose of 138 mg/dL. The Freestyle LibrePro professional CGM was used to investigate the reliability of the patient's SMBG and showed an average glucose of 165 mg/dL. An investigation into potential causes for falsely elevated HbA1c revealed a previously undiagnosed HbC trait. CONCLUSION: CGM is a valuable tool to assess HbA1c and SMBG discordance and to guide subsequent diabetes management. Our results suggest that the HbA1c may have been overestimated in our patient due to HbC trait; therefore, results must be interpreted with caution.

Frequency of Somatic TP53 Mutations in Combination with Known Pathogenic Mutations in Colon Adenocarcinoma, Non-Small Cell Lung Carcinoma, and Gliomas as Identified by Next-Generation Sequencing.

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancer. It encodes p53, a DNA-binding transcription factor that regulates multiple genes involved in DNA repair, metabolism, cell cycle arrest, apoptosis, and senescence. TP53 is associated with human cancer by mutations that lead to a loss of wild-type p53 function as well as mutations that confer alternate oncogenic functions that enable them to promote invasion, metastasis, proliferation, and cell survival. Identifying the discrete TP53 mutations in tumor cells may help direct therapies that are more effective. In this study, we identified the frequency of individual TP53 mutations in patients with colon adenocarcinoma (48%), non-small cell lung carcinoma (NSCLC) (36%), and glioma/glioblastoma (28%) at our institution using next-generation sequencing. We also identified the occurrence of somatic mutations in numerous actionable genes including BRAF, EGFR, KRAS, IDH1, and PIK3CA that occurred concurrently with these TP53 mutations. Of the 480 tumors examined that contained one or more mutations in the TP53 gene, 219 were colon adenocarcinomas, 215 were NSCLCs, and 46 were gliomas/glioblastomas. Among the patients positive for TP53 mutations diagnosed with colon adenocarcinoma, 50% also showed at least one mutation in pathogenic genes of which 14% were BRAF, 33% were KRAS, and 3% were NRAS. Forty-seven percent of NSCLC patients harboring TP53 mutations also had a mutation in at least one actionable pathogenic variant with the following frequencies: BRAF: 4%, EGFR: 10%, KRAS: 28%, and PIK3CA: 4%. Fifty-two percent of patients diagnosed with glioma/glioblastoma with a positive TP53 mutation had at least one concurrent mutation in a known pathogenic gene of which 9% were CDKN2A, 41% were IDH1, and 11% were PIK3CA.

Low serum alkaline phosphatase activity due to asymptomatic hypophosphatasia in a teenage girl.

OBJECTIVE: The case report details an unusual presentation of a teenage patient with hypophosphatasia. PATIENT AND METHODS: A 17 year-old female patient presented to endocrinology for the evaluation of fatigue and possible adrenal insufficiency. In the course of her clinical evaluation she was noted to have a low serum alkaline phosphatase activity. Relatively few conditions are associated with a low serum alkaline phosphatase including Wilson's disease, hypophosphatasia, pernicious anemia and untreated hypothyroidism. RESULTS: Laboratory testing for hypothyroidism were unrevealing, as were the results for vitamin B12 and vitamin D. Testing for Wilson's disease revealed a ceruloplasmin concentration of 165 mg/L (Reference Interval, 160-450 mg/L), however sequencing of the ATP7B gene revealed no deleterious mutations. Measurement of serum pyridoxal phosphate and urine phosphoethanolamine for the diagnosis of hypophosphatasia revealed concentrations of 541.5 nmol/L (reference interval: 29.6-295.5) and 707 mmol/mol creatinine (reference interval: <778 mmol/mol creatinine), respectively, consistent with a diagnosis of hypophosphatasia. CONCLUSIONS: Hypophosphatasia was initially considered an unlikely diagnosis for this patient given her lack of characteristic skeletal abnormalities. This diagnosis of hypophosphatasia in this case was complicated by a serum ceruloplasmin concentration at the lower end of the reference interval leading to the genetic testing for Wilson's disease.

Reference intervals and diagnostic ranges for serum free κ and free λ immunoglobulin light chains vary by instrument platform: Implications for classification of patient results in a multi-center study.

BACKGROUND: Serum free light chain (FLC) immunoglobulins are key biomarkers that aid in the diagnosis, prognosis and assessment of treatment response in patients with plasma cell disorders (PCD). Here we investigated the transference of manufacturer's reported κFLC, λFLC and κ to λ FLC reference intervals (RI) and established de novo FLC RI and diagnostic ranges on four instruments at three academic medical centers. In addition, we also compared the classification of patient FLC results using manufacturer's versus established RIs and diagnostic ranges. METHODS: CLSI EP28-A3C protocol was applied to investigate transference and establishment of FLC reference intervals on the cobas (Roche), Immage (Beckman), Optilite and SPA Plus (Binding Site). Serum κ FLC and λ FLC were measured in reference sera (N = 126) with estimation of central 95% RIs and FLC ratio diagnostic range (total range). Frequencies (%) in patient FLC results (N > 380 per institution) classified above, below or within manufacturer's versus established FLC RI were compared. RESULTS: Three of four instrument platforms did not exhibit acceptable transference of manufacturer's reported κ FLC RI. The manufacturer's reported FLC total diagnostic range did not encompass all values observed in reference sera for any of the four platforms evaluated. Established FLC ratio diagnostic ranges reduced the frequency of patient results classified above range for three of four platforms evaluated. CONCLUSIONS: Transference of manufacturer's reported FLC RIs may be inappropriate for select instrument platforms. De novo establishment of FLC RIs specific to instrument platform is highly recommended in order to assure correct patient result classification.

Method Validation of Human Chorionic Gonadotropin and α-Fetoprotein in Cerebrospinal Fluid: Aiding the Diagnosis of Intracranial Germ Cell Tumors.

BACKGROUND: Our study objective was to validate 2 individual methods to measure α-fetoprotein (AFP) and human chorionic gonadotropin (hCG) in cerebrospinal fluid (CSF) on the Roche cobas® 6000 analyzer. A 3-year retrospective chart review of CSF samples analyzed for AFP and hCG was also conducted. METHODS: Serum samples with high concentrations of AFP or hCG were added to aliquots of pooled CSF. Precision, linearity, detection limit, recovery, carryover, stability, and interference studies of the AFP and hCG+ß assays were performed. RESULTS: Within-day and day-to-day assay imprecision for AFP and hCG assays were <5% at all concentrations tested. The linear range of the AFP assay was established as 1.0-1100 µg/L, and limit of quantification (LOQ) was <1.0 µg/L. The linear range of the hCG assay was established as 1.0-9500 IU/L and LOQ 0.7 IU/L. There was no demonstrable matrix effect, and neither assay was affected by the presence of hemolysis or xanthochromia. AFP in CSF was stable at room and refrigerated temperatures for up to 48 h at concentrations of 19 and 306 µg/L but increased by 24 h at a concentration of 908 µg/L. AFP in CSF was stable frozen (-20 °C) for up to 7 days. hCG in CSF at all concentrations tested was stable at room, refrigerated, and frozen temperatures for up to 7 days. CONCLUSIONS: The Roche cobas 6000 AFP and hCG+ß assays accurately quantify AFP and hCG in CSF, facilitating rapid and accurate diagnosis as well as monitoring of intracranial germ cell tumors.