Evaluation of the stability of fecal immunochemical test specimens.

Fecal immunochemical tests (FIT) are used to screen for colorectal cancer by detecting blood present in stool. Patients collect FIT specimens at home in a sampling kit and return them to the lab for testing. At our institution, patients are instructed to return their specimens to the laboratory within seven days from collection, which is shorter than the manufacturer stated room temperature (RT) stability of 15 days. The objective of this study was to assess and verify the stability of FIT specimens at RT and to determine if refrigerated storage improves stability. A series of experiments were performed with the OC-Sensor DIANA iFOB Test system between 2017 and 2019, using a positive clinical cut-off of 75 ng/mL (15 µg/g) hemoglobin (Hb). Specimens were collected and categorized based on their initially measured Hb concentration and had repeated measurements for up to 21 days following collection. FIT specimens were stored either at RT or refrigerated. Our results show that FIT specimens have reduced concentrations of Hb compared to baseline when stored at RT; refrigeration improved FIT specimen stability but did not completely prevent the reduction in Hb concentration. Additionally, specimens marginally above the cut-off (initial concentrations between 75 and 100 ng/mL (15-20 µg/g)) that were stored at RT showed 100% positivity on the day of collection (n=33), 63% on Day 3 (n=19), 46% on Days 4/5 (n=26), and 38% on Days 6/7 (n=26). Finally, specimens with Hb values near the clinical cut-off appear to be particularly susceptible to false negatives as a result of the reduction in Hb over time. Therefore, laboratories should verify the specifics of their FIT tests before offering it to patients to reduce false negatives.

Transformation of Sequential Hospital and Outpatient Laboratory Data into Between-Day Reference Change Values.

BACKGROUND: Serial differences between intrapatient consecutive measurements can be transformed into Taylor series of variation vs time with the intersection at time = 0 (y0) equal to the total variation (analytical + biological + preanalytical). With small preanalytical variation, y0, expressed as a percentage of the mean, is equal to the variable component of the reference change value (RCV) calculation: (CVA2 + CVI2)1/2. METHODS: We determined the between-day RCV of patient data for 17 analytes and compared them to healthy participants' RCVs. We analyzed 653 consecutive days of Dartmouth-Hitchcock Roche Modular general chemistry data (4.2 million results: 60% inpatient, 40% outpatient). The serial patient values of 17 analytes were transformed into 95% 2-sided RCV (RCVAlternate), and 3 sets of RCVhealthy were calculated from 3 Roche Modular analyzers' quality control summaries and CVI derived from biological variation (BV) studies using healthy participants. RESULTS: The RCVAlternate values are similar to RCVhealthy derived from known components of variation. For sodium, chloride, bicarbonate calcium, magnesium, phosphate, alanine aminotransferase, albumin, and total protein, the RCVs are equivalent. As expected, increased variation was found for glucose, aspartate aminotransferase, creatinine, and potassium. Direct bilirubin and urea demonstrated lower variation. CONCLUSIONS: Our RCVAlternate values integrate known and unknown components of analytic, biologic, and preanalytic variation, and depict the variations observed by clinical teams that make medical decisions based on the test values. The RCVAlternate values are similar to the RCVhealthy values derived from known components of variation and suggest further studies to better understand the results being generated on actual patients tested in typical laboratory environments.

Measuring steroids from dried blood spots using tandem mass spectrometry to diagnose congenital adrenal hyperplasia.

BACKGROUND: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders that occur due to defects in the steroidogenesis pathway. Approximately 90% of CAH cases can be diagnosed by the measurement of serum 17-hydroxyprogesterone alone. However, the quantification of six additional steroids could significantly improve CAH laboratory diagnosis. Using dried blood spot (DBS) as specimen of choice can further improve patient care due to the small sample volume required for CAH diagnosis in neonates. METHODS: An optimized DBS sample preparation method was employed for steroids quantification without the need of derivatization. A LC-MS/MS assay was developed and optimized using a reverse phase-ultra high-pressure liquid chromatography (RP-UHPLC) system combined with triple quadrupole mass spectrometry using positive electrospray ionization mode. RESULTS: The assay was validated according to CLSI analytical guidelines, including lower limit of quantification (LLOQ), linearity, precision, accuracy, carryover, and method comparison. The analytical measuring range of the method for all steroids was 2.5, 5, or 10 ng/ml to 250 ng/ml in DBS, r2 ≥ 0.995. The LLOQ, intra-day and inter-day precision were 0.11-1.8 ng/ml, 1.2-6.4 ng/ml, 1.8-11.5%, and 5.3-13.8%, respectively. CONCLUSIONS: Our LC-MS/MS assay simultaneously detects 7 steroids for the diagnosis of CAH and can be readily implemented in clinical laboratories to provide superior analytical performance over traditional immunoassays.

A high-throughput test for diabetes care: An evaluation of the next generation Roche Cobas c 513 hemoglobin A1C assay.

OBJECTIVES: The level of glycated hemoglobin A (HbA1C) in blood is the preferred marker for diabetes monitoring and treatment. Here, we evaluate the analytical performance of the Roche Diagnostics Cobas c 513, a stand-alone HbA1C immunoassay analyzer. DESIGN AND METHODS: Performance was assessed with regards to imprecision, accuracy, linearity, method comparison against the Roche Cobas Integra 800 CTS, specimen stability, interference from common hemoglobin variants and hemoglobin F, and throughput. RESULTS: Within-run and between-run precisions were 0.5-0.7 and 0.8-1.3%CV, respectively. An average bias of -1.6% to proficiency survey samples was observed. The c 513 correlated well with the Integra (slope = 0.94, y-intercept = 0.50, and correlation coefficient = 0.998). The effect of hemoglobin variants on this assay was negligible while specimens containing ≥10% HbF demonstrated a negative bias. The c 513 instrument can process up to 340 samples per hour. CONCLUSIONS: The c 513 is a precise, accurate, automated high throughput analyzer for measuring HbA1C in large laboratories.

Strategies for mitigating risk posed by biotin interference on clinical immunoassays.

Recently, biotin use as an oral supplement has increased significantly among the general population. Biotin is a water soluble B-vitamin and is marketed to improve the cosmetic appearance of hair, skin, and nails. In addition, high-dose biotin (>5 mg/day) is prescribed to treat inborn errors of metabolism and multiple sclerosis. Many commercial immunoassays employ the high affinity interaction between biotin and streptavidin, a protein purified from bacteria, as part of the analyte capture mechanism. As such, these immunoassays are subject to this interference. The list of affected immunoassays is vendor specific but includes tests for troponin, serum and urine Beta hCG, thyroid function, and tumor markers. The interference can be positive or negative in nature depending on the immunoassay. To address this issue, patients are recommended to abstain from taking biotin supplements for 48 h, and laboratorians and clinicians must be familiar with the potential for biotin interference in performed lab tests. Here we describe strategies to treat high biotin specimens and make them suitable for testing; and detail a number of approaches used successfully by our laboratory to educate patients, doctors, and other healthcare professionals about this interference and to mitigate the posed patient safety risk.

Intervention to Reduce Unnecessary Glucose Tolerance Testing in Pregnant Women.

BACKGROUND: Gestational diabetes mellitus (GDM) can be diagnosed in pregnant women by increased fasting plasma glucose alone, which eliminates the need for performing a 75 g oral glucose tolerance test (OGTT). If whole blood glucose meters are used to triage fasting samples in order to decide whether to give the glucose drink, a cutpoint with appropriate sensitivity and specificity for elevated fasting plasma glucose is needed. METHODS: The number of GDM diagnoses by increased fasting plasma glucose alone was determined from specimens collected and tested at core laboratories in urban hospitals, rural health centers, and from specimens collected at patient phlebotomy service centers (PSCs) for plasma testing at a central laboratory. The number of glucose drinks avoided was counted after implementing the diagnostic cutoff of ≥95 mg/dL (5.3 mmol/L) at urban hospitals and rural health centers, which have on-site plasma testing, and after selecting a PSC meter fasting venous whole blood glucose cutpoint after calculating sensitivity and specificity for plasma glucose ≥95 mg/dL (5.3 mmol/L) using logistic regression. RESULTS: Among 4850 OGTTs, there were 1315 GDM diagnoses annually, of which 409 were from increased fasting plasma glucose. Ninety-one percent of OGTTs were performed at PSCs. If a fasting plasma glucose cutpoint of ≥95 mg/dL (5.3 mmol/L) was implemented at urban hospitals and rural health centers and a meter fasting venous whole blood glucose cutpoint of ≥108 mg/dL (6.0 mmol/L) (25% sensitivity, 99.9% specificity) was implemented at PSCs, the drink would be appropriately avoided by 145 patients/year, and inappropriately avoided by 3 patients/year. After implementing these cutpoints, the drink was appropriately avoided in 91 patients during a 36-week period, with none inappropriately avoiding it. CONCLUSION: Modifying fasting glucose cutpoints reduced unnecessary diagnostic OGTTs in pregnant women.

Elimination of 72-Hour Quantitative Fecal Fat Testing by Restriction, Laboratory Consultation, and Evaluation of Specimen Weight and Fat Globules.

BACKGROUND: The 72-h quantitative fecal fat test has been mostly obsolete for many years. Our objective was to reduce and eliminate the use of this test, while providing suitable alternatives. METHODS: We assessed (2010-2016) utilization of the fecal fat test in Calgary, Central Alberta, and Southern Alberta, Canada. Alternatives were identified through literature review and consultation with gastroenterologist stakeholders. Logistic regression and ROC curves were used to characterize discrimination power of 72-h specimen weight on abnormal fat excretion. This was also examined in 91 subspecimens that were additionally tested for the presence of fat globules. RESULTS: As 69% of fecal fat tests (total, 106/year) were on adults (age ≥ 18), stakeholders agreed that adult specimens should not be tested until ordering physicians consulted with a clinical biochemist. This change reduced fecal fat testing by 81% to 20/year in 2015. The 72-h specimen weight was a significant predictor of abnormal fat excretion [P < 0.001; area under curve (AUC) = 0.75-0.79, n = 115-417] in historic fecal fat data. A similar result was observed among subspecimens (AUC = 0.70), which improved when additionally considering the presence of fat globules (AUC = 0.74). Stakeholders consented to replacing fecal fat with a comparison of specimen weight to cutpoints with 80% specificity for abnormal fat excretion, and the test for fat globules. CONCLUSION: Through stakeholder engagement, we implemented changes that eliminated 72-h quantitative fecal fat testing in a large geographic region in Alberta, Canada. Future fecal fat orders would be reflexed to an assessment of 72-h specimen weight and a qualitative test for fat globules in stool.

The effect of adverse intrauterine conditions, early childhood growth and famine exposure on age at menopause: a systematic review.

When the follicle reserve, which is developed solely during the fetal period, is depleted, women enter menopause. Intrauterine and childhood adverse conditions might affect the ovarian capacity by influencing follicle production in the first trimester, limiting the initial follicle pool or mediate an accelerated follicular loss thereafter. To investigate if adverse early life influences result in younger age at menopause, the following online databases were systematically searched: PubMed, EMBASE, CINHAL (EBSCO) and Cochrane library (Wiley) up to 1 January 2017. Eligibility, data extraction and quality assessment was independently performed by two researchers. A total of 5278 studies were identified, 11 studies were deemed eligible and included. Nine were cohort studies, 1 case-control study and 1 twin study. Due to the diversity of reported data and risk estimates we were unable to pool data or perform meta-analysis on pooled data. Prenatal and childhood exposure to famine was significantly associated to an earlier age at menopause in three studies. Mean differences in age at menopause varied from 4 months up to 1.7 years between famine exposed and unexposed women. Three studies described a significant association between a low weight at ages 1 or 2 and a younger age at menopause. A younger age at menopause was associated with a higher weight at birth in only one study and with a high ponderal index, a measure for fatness at birth in another study. None of the nine studies reporting on low birth weight and age at natural menopause find a significant association.

Influence of diurnal variation and fasting on serum iron concentrations in a community-based population.

OBJECTIVES: Serum iron is an important clinical test to help identify cases of iron deficiency or overload. Fluctuations caused by diurnal variation and diet are thought to influence test results, which may affect clinical patient management. We examined the impact of these preanalytical factors on iron concentrations in a large community-based cohort. DESIGN AND METHODS: Serum iron concentration, blood collection time, fasting duration, patient age and sex were obtained for community-based clinical testing from the Laboratory Information Service at Calgary Laboratory Services for the period of January 2011 to December 2015. RESULTS: A total of 276,307 individual test results were obtained. Iron levels were relatively high over a long period from 8:00 to 15:00. Mean concentrations were highest at blood collection times of 11:00 for adult men and 12:00 for adult women and children, however iron levels peaked as late as 15:00 in teenagers. With regard to fasting, iron levels required approximately 5h post-prandial time to return to a baseline, except for children and teenage females where no significant variation was seen until after 11h fasting. After 10h fasting, iron concentrations in all patient groups gradually increased to higher levels compared to earlier fasting times. CONCLUSIONS: Serum iron concentrations remain reasonably stable during most daytime hours for testing purposes. In adults, blood collection after 5 to 9h fasting provides a representative estimate of a patient's iron levels. For patients who have fasted overnight, i.e. ≥12h fasting, clinicians should be aware that iron concentrations may be elevated beyond otherwise usual levels.

Premature ovarian insufficiency and perinatal parameters: A retrospective case-control study.

OBJECTIVE: The peak number of oocytes is reached during intrauterine development, after which numbers decline until reserves are depleted and a woman enters menopause. In premature ovarian insufficiency (POI), the process of follicle depletion occurs at a young age (generally taken as 40 years); the condition affects about 1% of women. In this study, we investigate whether women with POI had experienced a different perinatal milieu, as reflected in their birth weight or prematurity. STUDY DESIGN: In this retrospective case-control study, we evaluated whether women diagnosed with POI had a different birth weight or prematurity rate (<37 weeks) compared with women aged over 40 at natural menopause (the controls). Binary logistic regression models were used to analyze the data and correct for smoking. 59 women with POI and 92 controls were recruited. RESULTS: 13.6% of women diagnosed with POI were born prematurely, compared with 6.6% of controls (p=0.018). Corrected for gestational age, women with POI did not have a different birth weight compared with controls. As a consequence of the design of our study, mean age at time of interview differed significantly between groups, at 37.5 and 46 years respectively for women diagnosed with POI and controls. Years of oral contraception use, smoking, age at menarche, BMI and education levels were similar in the two groups. CONCLUSION: Our findings indicate that prematurity is a risk factor for POI. Prenatal factors contributing to prematurity, or postnatal factors that are the result of prematurity, may lead to early follicle depletion.

Birthweight and PCOS: systematic review and meta-analysis.

STUDY QUESTION: Are intrauterine conditions, reflected in birthweight, associated with the development of polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Our study indicates that a low birthweight as a summary measure of intrauterine environment may be associated with PCOS when diagnosed according to the Rotterdam criteria. WHAT IS ALREADY KNOWN: The etiology of PCOS is still largely unknown. Besides subfertility, women diagnosed with PCOS have an increased risk of chronic health issues. PCOS has been linked to adverse prenatal conditions, including a low birthweight. STUDY DESIGN SIZE DURATION: A systematic search of the literature and meta-analysis of pooled data was undertaken, according to the preferred reporting items for systematic reviews and meta-analysis (PRISMA) and meta-analysis of observational studies in epidemiology (MOOSE) guidelines. PARTICIPANTS/MATERIALS SETTING METHOD: The following online databases were systematically searched: PubMed, EMBASE, CINAHL (via EBSCO) and Cochrane library up to 10 June 2017, with no language or date restrictions. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 1484 studies were identified of which 16 met the inclusion criteria and 14 provided data for meta-analysis. The exposure variable birthweight was either analyzed as a categorical variable using the birthweight categories <2.5, 2.5-4 and >4 kg, or as a continuous variable. We composed a birthweight category consisting of birthweights <2.5 kg plus birthweights >4 kg, reflecting extreme birthweights. In a subset analysis, we investigated the association between a low birthweight and PCOS while differentiating between Rotterdam and NIH criteria. When diagnosed according to the Rotterdam criteria, women born with birthweights lower than 2.5 kg had an odds ratio [95% CI] of 1.76 [1.14,2.70] for PCOS compared to women born with birthweights higher than 2.5 kg. For the latter analysis, we were able include 1252 women (I 2 = 16%). There was no significant effect of birthweight on PCOS when diagnosed according to NIH criteria. LIMITATIONS REASONS FOR CAUTION: The funnel plot of the studies providing data for the meta-analysis and the subset analysis indicates a publication bias. WIDER IMPLICATIONS OF THE FINDINGS: A low birthweight could be a risk factor for PCOS when diagnosed according to the Rotterdam criteria. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: The protocol of this study was registered at PROSPERO under registration number CRD42016048972.

Fasting time and vitamin B12 levels in a community-based population.

OBJECTIVES: Vitamin B12, also known as cobalamin (Cbl), is an essential vitamin that manifests with numerous severe but non-specific symptoms in cases of deficiency. Assessing Cbl status often requires fasting, although this requirement is not standard between institutions. This study evaluated the impact of fasting on Cbl levels in a large community-based cohort in an effort to promote standardization of Cbl testing between sites. DESIGN AND METHODS: Laboratory data for Cbl, fasting time, patient age and sex were obtained from laboratory information service from Calgary Laboratory Services (CLS) for the period of April 2011 to June 2015. CLS is the sole supplier of laboratory services in the Southern Alberta region in Canada (population, approximately 1.4 million). To investigate potential sex-specific effects of fasting on Cbl levels, males and females were analyzed separately using linear regression models. RESULTS: A total of 346,957 individual patient results (196,849 females, 146,085 males) were obtained. The mean plasma Cbl level was 386.5 (±195.6) pmol/L and 412.0 (±220.8) pmol/L for males and females, respectively. Linear regression analysis showed fasting had no significant association with Cbl levels in females; however a statistically significant decrease of 0.9pmol/L/hour fasting (p<0.001) was noted in males. CONCLUSIONS: The broad population variance in Cbl suggests the slight gender-specific differences noted in this study are insignificant. Despite this, fasting has the potential to contribute to higher rates of Cbl deficiency in men. Together, these data suggest fasting should be excluded as a requirement for evaluating plasma Cbl.

Sensitivity and Specificity of Community Fecal Immunotesting Screening for Colorectal Carcinoma in a High-Risk Canadian Population.

CONTEXT: Community-based programs are a common way of promoting colorectal cancer screening by primary care physicians. Fecal immunochemical testing (FIT) is a screening method commonly used in such programs. Fecal immunochemical testing has advantages to the patient as well as to clinical laboratories. OBJECTIVE: To assess the operational test characteristics of a FIT pilot program in Calgary, Alberta, Canada, between April 2011 and May 2012. DESIGN: Four hundred fifty-seven high-risk patients undergoing both FIT and colonoscopy were included. Areas under the curve and positive predictive values were derived for FIT values and biopsy-proven neoplasia. Subgroup analysis was also performed on men and women and for ages older and younger than the mean age of 62 years. RESULTS: For colorectal carcinoma and colonic adenomas the areas under the curve were 0.79 (95% confidence interval 0.71-0.87) and 0.60 (95% confidence interval 0.54-0.65), respectively. The positive predictive value of a positive FIT result for any neoplasia was 53%. The overall performance of the test for all neoplasia was better for men and better for older individuals. CONCLUSIONS: The performance of FIT in this clinical setting was very good for detecting carcinoma, but marginal for detection of colonic adenomas.

Sociodemographic correlates of fecal immunotesting for colorectal cancer screening.

OBJECTIVES: Community fecal immunochemical testing (FIT) screening programs are important for detecting early disease and can be effective when promoted by primary care physicians. Screening rates remain low across Canada and may be associated with sociodemographic factors. DESIGN AND METHODS: Fecal immunochemical testing results for a new community-based screening program were obtained from Calgary Laboratory Services from November 18, 2013 to May 31, 2014. Screening rates were determined for specific age and sex cohorts and sociodemographic factors were inferred from census Canada data. Poisson regression and generalized estimating equations were used to test associations of screening rate with sociodemographic variables. RESULTS: A total of 27,572 results were screened and included in our analysis. Recent immigrants (RR = 0.18, P = <.0001), Aboriginal First Nations (RR = 0.39, P = 0.01), Aboriginal Metis (RR = 0.14, P = 0.0003), visible minority Black (RR = 0.35, P = 0.0002), and higher education (RR = 0.65, P = <.0001) were associated with decreased screening. Visible minority Chinese (RR = 1.72, P = <.0001) were more likely to be screened. Household income was not associated with screening rate. Older individuals and females were less likely to be screened. CONCLUSION: There is significant geographic variation in screening rates in Calgary. These are associated with a number of sociodemographic factors.

Simultaneous detection of 19 drugs of abuse on dried urine spot by liquid chromatography-tandem mass spectrometry.

BACKGROUND: The lack of specificity of immunoassays for drugs of abuse testing (DAT), and concerns over its cost in conjunction with reflex confirmatory tests prompted us to investigate the combinatorial use of dried urine spot (DUS) and LC-MS/MS as an alternative. METHODS: The method development and validation were performed in accordance with the guidelines published by FDA and CLSI. RESULTS: In this study we established and validated the precision, accuracy, and linearity of our DUS-LC-MS/MS method, and assessed the recovery, interference, and carryover as well. The linearity check for all 19 analytes demonstrated slopes between 0.94 and 1.04, and R(2) always greater than 0.99. Between-batch CV for QC at 4 difference levels ranged from 1.1% to 10%, where CV of LLOQ ranged from 1.2% to 12.8% and CV of ULOQ ranged from 0.8% to 5.1%. A concordance study with patient specimens between our method and GC-MS demonstrated 80.8% to 100% agreement. Stability of DUS specimens was assessed up to 30 days and the measured concentrations ranged from 94% to 114% of the 100 ng/mL urine calibrator used for this assessment. CONCLUSIONS: We established and validated a DUS-LC-MS/MS method for DAT that conforms to the guidelines dictated by FDA, CLSI, and SAMHSA. While our method with high sensitivity and specificity provides an alternative diagnostic utility to EMIT immunoassays, it also offers superior solutions in specimen transportation, preservation, and storage. The benefits of our method are apparent in reducing turnaround time and test costs that result in better patient care.

Haptoglobin phenotype and apolipoprotein E polymorphism: relationship to posttraumatic seizures and neuropsychological functioning after traumatic brain injury.

The relationship of genetic predisposition to reduced iron capacity and apolipoprotein E (APOE) to posttraumatic seizures (PTSs) and neuropsychological outcomes was investigated in patients with traumatic brain injuries from a prior valproate clinical study. Haptoglobin concentration/phenotype and APOE genotype were determined in 25 patients with PTSs and 26 control (no PTSs) subjects approximately 10 years after traumatic brain injury. Haptoglobin phenotype was also determined in previously collected frozen samples for 25 additional patients with PTSs and 32 no-PTS subjects. There was no relationship between haptoglobin phenotype or APOE genotype and occurrence of PTSs. APOE genotype was not related to neuropsychological outcome; however, when adjustments were made for differences in educational levels, APOE epsilon4 subjects did worse, especially on tests of verbal intellectual and verbal memory skills. In contrast to our hypothesis, those with haptoglobin 1-1 (high-affinity binder of hemoglobin) scored somewhat worse on Verbal IQ and Tapping D at 1 and 12 months after injury.