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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.

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.

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.

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).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.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 value53.50 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 value30.5 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 value436 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.1250AG (p.Asn417Ser), c.1133AT (p.Asp378Val), c.881AC (p.Asp294Ala), c.1171CT (p.Arg391Cys), and c.571GA, (p.Glu191Lys).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.

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.

Hypophosphatasia (HPP) is a rare disease caused by a loss-of-function mutation in the ALPL gene leading to a deficiency in the tissue-non-specific isoenzyme alkaline phosphatase (ALP) and excess of extracellular inorganic pyrophosphate (PPi) and pyridoxal 5’phosphate (PLP). Patients with HPP have a widely variable clinical phenotype, from neonatal seizures and hypomineralization to isolated dental or joint disease in adults. Patients present with ALP activity levels below their age-adjusted reference interval and elevations in PLP. This variable clinical phenotype leads to significant diagnostic challenges, particularly in those with minor disease manifestations. Biomarkers for diagnosing HPP and monitoring patients on enzyme replacement therapy (ERT) are limited. Low ALP activity is not specific for HPP; it can be low in other conditions including untreated hypothyroidism, and other skeletal dysplasias. Accurate PLP assessment is limited in patients on ERT due to hydrolysis of the substrate. Phosphoethanolamine (PEA) is a substrate hydrolyzed by TNSALP and elevated levels of PEA may be observed in HPP, supporting the diagnosis of HPP, but have been reported to be non-specific. We hypothesized that urine PEA levels could be used to diagnose HPP and as a surrogate marker for ERT compliance. We performed a retrospective analysis on 83 adult patients (63F: 20M) presenting for investigation or follow-up of HPP from 7/1/2014 to 1/7/2020. Urine was sent to the Children’s Hospital in Colorado for PEA testing. Patients were diagnosed with HPP if they had ALP levels persistently below the age-adjusted reference interval, were positive for an ALPL gene mutation/had a family history of HPP and had musculoskeletal/dental symptoms consistent with the disease. Patients were negative for HPP if ALP was not persistently low, vitamin B6 levels were normal without supplementation, negative ALPL genetic testing results (when available) and no musculoskeletal/dental symptoms. PEA levels were not considered for diagnosis. The following was collected from the EMR: PEA results, diagnosis, and ERT status. PEA levels were higher in patients with HPP not on ERT (median=117.0, N=39) vs. those negative for HPP (median=24.0, N=15, p53.5 ng/ml (sensitivity=79.5%) with an NPV of 65.2% and PPV of 100%. Initial PEA values in patients with HPP and not on ERT were higher (median 117.0, N=39) than patients on ERT (median 65.0, N=16, p=0.004). Patients who began ERT had a decline in PEA levels after treatment (mean decrease 68.1%). PEA is a specific diagnostic marker for HPP in patients undergoing investigation for HPP and may be used as a surrogate marker to monitor ERT compliance. Future studies are necessary to evaluate the association between PEA levels and functional performance.

Introduction: Hypophosphatasia (HPP) is an inborn error of metabolism due to deficiency of tissue non-specific alkaline phosphatase (TNSALP), characterized by a wide range of skeletal and neurological symptoms. Little is known about the mechanism of neurologic involvement, but it may result from inefficient hydrolysis of vitamin B6 (pyridoxal 5’-phosphate, PLP), which is involved in neurotransmitter synthesis and is markedly elevated in HPP. However, it remains unknown what PLP levels are in the cerebrospinal fluid (CSF) of patients with HPP. We report two cases: (1) one with pre- and post-treatment CSF PLP levels and (2) one with pre-treatment CSF PLP and neurotransmitter levels. Case 1: A 30-year-old woman with extensive fracture history presented with seizure-like activity. Laboratory evaluation while on no B6 supplementation was notable for low ALP (14 U/L, ref: 40-150), high plasma PLP (362 mcg/L, ref: 5-50), and high CSF PLP (16 mcg/L), with a low CSF/plasma PLP ratio (0.04, ref: 0.1-0.7[1]). Genetic testing showed a pathogenic variant of TNSALP (Het c.1133A>T, p.Asp378Val, rs 121918008). After initiation of enzyme replacement therapy (ERT), her plasma and CSF PLP levels normalized to 6.3 and 1.4 mcg/L, respectively, with a ratio of 0.23. She reported improvement in energy level and seizure-like activity after several months of ERT. Case 2: A 34-year-old man with no history of skeletal pathology presented with diffuse musculoskeletal pain, weakness, and loss of sensory function attributed to generalized peripheral neuropathy. Laboratory evaluation while on no B6 supplementation was notable for low ALP (23 U/L), high plasma PLP (496 mcg/L), and high CSF PLP (17 mcg/L) with a low CSF/plasma ratio (0.03). CSF neurotransmitter levels were also low, including 5-hydroxyindoleacetic acid (40 nmol/L, ref: 67-140) and homovanillic acid (115 nmol/L, ref: 145-324). Genetic testing showed heterozygous expression of a catastrophic frameshift mutation of TNSALP (c.662del, p.Gly221Valfs*56). ERT is being considered at this time. Conclusions: We report the novel findings that in these patients with HPP: 1. CSF PLP levels are markedly elevated, though to a much lesser extent than plasma levels. Elevated CSF/plasma ratios suggest that transport of PLP and its metabolites across the blood brain barrier may be impaired in HPP. 2. Decreased CSF neurotransmitter levels, as seen in our patient, may contribute to neurologic symptoms of HPP. 3. Enzyme replacement therapy normalized both CSF and plasma PLP in case 1. Further studies are needed to understand how these changes affect patient symptoms and prognosis. Citation: 1. Albersen, M., et al., Vitamin B-6 vitamers in human plasma and cerebrospinal fluid. Am J Clin Nutr, 2014. 100(2): p. 587-92

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.

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.

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+β assays were performed. Results Within-day and day-to-day assay imprecision for AFP and hCG assays were Conclusions The Roche cobas 6000 AFP and hCG+β assays accurately quantify AFP and hCG in CSF, facilitating rapid and accurate diagnosis as well as monitoring of intracranial germ cell tumors.

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 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.

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

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.Abbreviations: CGM = continuous glucose monitoring; HbA1c = hemoglobin A1c; HbC = hemoglobin C; HPLC = high-performance liquid chromatography; POC = point-of-care; SMBG = self-monitored blood glucose

A 33-year-old white man with a history of alcohol abuse presented to the emergency department for voluntary alcohol detoxification several hours after consuming approximately 8–10 oz (237–296 mL) of rubbing alcohol (isopropanol). He complained of anxiety and nausea and had at least 1 episode of reported hematemesis. At times, the patient was tachycardic but remained hemodynamically stable with blood pressure between 120s–140s/70s–100s mmHg. Notable laboratory values on admission include an increased anion gap of 24 mEq/L (reference range, 0–12 mEq/L), and increased plasma creatinine concentration of 1.27 mg/dL (reference range, 0.72–1.25 mg/dL). The patient's baseline plasma creatinine concentration had previously ranged from 0.80 to 0.95 mg/dL, 6–12 months before this hospitalization. Trace amounts of urine ketones were detected. The patient's urine drug screen was positive for acetaminophen, and ethanol and presumptive positive for benzodiazepines. Volatile testing using gas chromatography with flame ionization detection yielded an ethanol concentration of

A 16-year-old African-American male with known sickle/β+ thalassemia disease (HbS/β thalassemia) presented to the hematology clinic for management. Hemoglobin (Hb) variant analysis was performed using the BioRad Variant II HPLC β-thalassemia short program, which revealed HbF (fetal Hb), HbA, HbA2, and HbS, as well as HbC, …

A blood gas sample (Fig. 1) was sent to the laboratory from a 64-year-old male patient with stage IV non–small-cell lung cancer and paraneoplastic dermatomyositis. The results for electrolytes, glucose, pH, pCO2, and pO2 were unremarkable, but the instrument could not generate co-oximetry (e.g., oxyhemoglobin) results. Despite extensive mixing before analysis, the erythrocytes …

A 34-year-old healthy man underwent oral surgery and returned multiple times thereafter with recurrent bleeding from the surgical site. Over the course of 1 month and several visits, the bleeding was stopped temporarily with sutures and cauterization but recurred after each intervention. He had a history of excessive bleeding after dental extractions and developed a hematoma after a vasectomy. There was …