Dina N. Greene

Limitations in qualitative point of care hCG tests for detecting early pregnancy.

BACKGROUNDnDetection of early pregnancy in the healthcare setting is important for proper patient management. Qualitative point-of-care (POC) human chorionic gonadotropin (hCG) testing is often used. There is a gap in the literature for evaluating patient samples with low (<300 IU/l) hCG concentration using POC devices.nnnMETHODSnSpecimens (urine, n=289; serum, n=269) with hCG concentrations between 2 and >5000 IU/l were evaluated by hCG POC testing. Approximately half of these had an hCG concentration<300 IU/l. All specimens were evaluated using the OSOM and QuickVue+ POC hCG devices. Pregnancy status was evaluated by patient chart review.nnnRESULTSnThe OSOM and QuickVue+ devices were able to detect hCG in 53% (OSOM) and 78% (QuickVue+) of urine samples with an hCG concentration range between 20 and 300 IU/l. Serum samples with an hCG concentration range between 10 and 300 IU/l gave positive results in 79 and 91% of samples tested using the OSOM and QuickVue+, respectively. False negative results could not be attributed to the high-dose hook effect, the hCG variant effect, or the interpreters evaluation. The mean gestational age for specimens corresponding to the false negative results was 4 weeks.nnnCONCLUSIONSnThe sensitivity of 2 commonly used hCG POC devices is insufficient for detecting very early pregnancy.

Advances in detection of hemoglobinopathies.

Hemoglobin disorders are recognized as one of the most common inherited diseases worldwide. Detecting and characterizing variant hemoglobins and thalassemias depends primarily on clinical laboratory methods. Multiple biophysical, biochemical, and genetic assays are available to provide phenotypic or genotypic evidence of pathology. For many years conventional slab-gel electrophoresis and HPLC were the most commonly utilized laboratory methods. However, the field has rapidly expanded to regularly include capillary zone electrophoresis, molecular assays, and, more recently, mass spectrometric assays. Interpretation of these techniques is, in general, complicated because of the involvement of multiple polymorphic genes. Proper characterization of hemoglobin variants is necessary for diagnosis, primary prevention and genetic counseling for underlying disorders. This review provides an overview of the current hemoglobin analysis techniques, and also discusses technologies that have potential to translate into widespread clinical settings.

Total Analytic error for low cardiac troponin concentrations (<10 ng/L) by use of a high-sensitivity cardiac troponin assay

To the Editor:nnThere are considerable data suggesting that low cardiac troponin (cTn)1 concentrations measurable only by high-sensitivity cTn (hs-cTn) assays can be used for medical decision-making (1). However, there are limited data on the long-term analytical performance of hs-cTn related to the bias and imprecision associated with cTn concentrations ≤10 ng/L, which are not measurable with contemporary assays (2). For quality assurance purposes having a total analytic error (TAE) criterion at low concentrations would help identify problematic hs-cTn reagent/calibrator lots and suboptimal analyzer performance. This is a practical concern, as previous negative biases with the Roche hs-cTnT assay were identified and ultimately corrected by the manufacturer, but only after multiple different reagent lots were used clinically (3). Lot-to-lot or batch-to-batch evaluations for reagents and calibrators to identify minor shifts in hs-cTn results can be difficult, time-consuming, and expensive. The goal of this analysis was to establish a realistic TAE for cTnI concentrations ≤10 ng/L measured by the Abbott assay by assessing concentration biases between different lots of reagents/calibrators and imprecision at low cTn concentrations.nnOur approach to evaluating new hs-cTn lots involved comparing approximately 10 samples (fresh, not frozen) with cTn concentrations that spanned the measuring range on both lots, ideally with 3 samples containing cTn ≤10 ng/L (4). We pooled results from lot-to-lot comparisons over a 2-year period (2015–2016), which allowed us to assess the long-term difference …

Age, sex, and racial influences on the Beckman Coulter AccuTnI+3 99th percentile.

BACKGROUNDnBeckman Coulter recently released a new cardiac troponin I (cTnI) assay, AccuTnI+3, for the Access 2 and DxI platforms. We validated the stated 99th percentile (20 ng/l) using a large population of healthy adults representative of the Northern California population.nnnMETHODSnWithin a large sample of healthy adult members receiving care at Kaiser Permanente, cTnI was quantified in residual specimens using the AccuTnI+3 assay. Patients were selected based on pre-defined criteria extracted from a comprehensive electronic medical record. All specimens with a cTnI concentration >30ng/l were repeated; specimens that had a reproducible result >30 ng/l were subject to heterophile blocking procedure. 99th percentiles were calculated based on age, sex, race and body mass index categories.nnnRESULTSnAmong 1764 tested subjects, the 99th percentile for all samples was 25 ng/l. Sex differences were observed; the male and female 99th percentiles were 31 and 21 ng/l, respectively (p=0.001). Age (range evaluated 18-89y, median 47y) also had a significant influence on the value (p=0.003), but there were no significant differences by race. False positive results were detected in 0.9% of specimens (0.6% fliers and 0.3% heterophile antibodies), corresponding to 52% of all results >30 ng/l.nnnCONCLUSIONSnAmong a large, representative cohort of healthy adults, we found a 99th percentile value consistent with prior studies based on highly selected small patient samples. Sex and age-specific upper reference limits for cTnI should be considered. In this cohort, about half the findings above the 99th percentile were false positives. Avoiding reporting erroneous results requires implementation of quality indicators.

Endogenous alkaline phosphatase interference in cardiac troponin I and other sensitive chemiluminescence immunoassays that use alkaline phosphatase activity for signal amplification.

BACKGROUNDnFalse positive cardiac troponin results can lead to inappropriate diagnosis. Our laboratory workflow includes systematic quality practices to identify false positive cardiac troponin I (cTnI) results reported by the DxI AccuTnI+3 assay, which uses alkaline phosphatase (ALP) for signal amplification. Recently, a sample with elevated cTnI failed our quality standards and was found to have extremely elevated endogenous ALP activity. The objective of this study was to determine the true cTnI concentration and evaluate whether ALP was the source of interference.nnnMETHODSnThe suspicious cTnI result was evaluated by repeat analyses, dilution, heterophile blocking treatment, alternative methodology (Vista), and heat treatment. Purified ALP was added to reference serum and we quantified DxI cTnI and human chorionic gonadotropin (hCG). Next, cTnI and/or hCG was measured in specimens with normal (N=20) or elevated (N=26) ALP using DxI and Vista assays. Finally, cTnI was quantified using a prototype, ALP-dependent high-sensitivity assay.nnnRESULTSnThe sentinel samples DxI-cTnI results were imprecise on repeat, linear on dilution, unaffected by heterophile blocking antibodies, and correlated with ALP lability following heat treatment. The Vista-cTnI concentrations were ~7-fold lower. Addition of purified ALP to reference serum linearly increased the DxI-cTnI results. DxI-hCG results also appeared affected by ALP. Several independent patients specimens with elevated ALP appeared to have falsely elevated DxI-cTnI and DxI-hCG.nnnCONCLUSIONSnElevated ALP can interfere with contemporary, ALP-dependent immunoassays, including DxI-cTnI and DxI-hCG. The validation of such methods should include evaluations for endogenous ALP interference. Specimens with ALP >1000U/L and elevated DxI-cTnI should be evaluated for ALP interference.

Association Between Laboratory Calibration of a Serum Bilirubin Assay, Neonatal Bilirubin Levels, and Phototherapy Use

IMPORTANCEnThe American Academy of Pediatrics treatment recommendations for neonatal jaundice are based on age-specific total serum bilirubin (TSB) levels. In May 2012, Ortho Clinical Diagnostics adjusted the calibrator values for Vitros Chemistry Products BuBc Slides (Ortho Clinical Diagnostics), a widely used method to quantify TSB, after concerns of positively biased results.nnnOBJECTIVEnTo investigate the association between recalibration of a reflectance spectrophotometry serum bilirubin assay and TSB levels and phototherapy use among newborns.nnnDESIGN, SETTING, AND PARTICIPANTSnDescriptive study comparing TSB levels and phototherapy use before and after recalibration at Kaiser Permanente Northern California, a large, integrated health care delivery system. The study evaluated live births at or after 35 weeks gestation at 12 facilities that used universal serum bilirubin screening before (January 1, 2010, through April 30, 2012; nu2009=u200961u202f677) and after (July 1, 2012, through December 31, 2013; nu2009=u200942u202f571) recalibration. The analysis took place in December 2015.nnnINTERVENTIONnRecalibration of bilirubin testing instruments.nnnMAIN OUTCOMES AND MEASURESnProportions of newborns with (1) at least 1 TSB value at or above 15 mg/dL; (2) at least 1 TSB level exceeding the American Academy of Pediatrics phototherapy threshold; (3) phototherapy during the birth hospitalization; and (4) at least 1 readmission for phototherapy.nnnRESULTSnIn 104u202f420 infants (61u202f677 in the prerecalibration period and 42u202f511 in the postrecalibration period), a TSB was obtained in 99.2% of infants during birth and in 99.5% of infants within the first 30 days after birth. The postrecalibration period was associated with a 1.25 mg/dL (95% CI, 1.19-1.31; P < .001) decrease in mean maximum TSB, which led to a 39% relative reduction (from 20.4% to 12.4%) in infants with a TSB level of 15 mg/dL or more and a 51% relative reduction (from 9.3% to 4.5%) in infants with a TSB level that was at or above the American Academy of Pediatrics phototherapy threshold. Phototherapy during birth hospitalizations was reduced by 59% (absolute risk reduction, 5.5%; 95% CI, 4.7%-6.1%) and readmissions for phototherapy by 53% (absolute risk reduction, 1.8%; 95% CI, 1.4%-2.3%).nnnCONCLUSIONS AND RELEVANCEnModest recalibration-induced reductions in mean TSB concentrations was associated with a significant reduction in the percentage of infants with clinically significant hyperbilirubinemia. Current laboratory accuracy standards are insufficient to detect biases that can have significant clinical effect. These data underline the need for increased integration of laboratory expertise into clinical guidelines and to support international initiatives to standardize laboratory measurements.

Falsely undetectable TSH in a cohort of South Asian euthyroid patients.

CONTEXTnAn index case of a clinically euthyroid woman of South Asian descent was identified with discordant TSH results: undetectable TSH on our routine assay and normal TSH on an alternate assay. Low TSH concentrations due to functionally compromising TSH mutations have been reported. Here we describe a new phenomenon of functional TSH that is undetectable by 4 widely used US Food and Drug Administration (FDA)-approved TSH immunoassays marketed by a single vendor.nnnOBJECTIVEnThe purpose of this study was to identify additional cases and investigate the cause of the falsely undetectable TSH.nnnDESIGNnAll samples with TSH results of <0.01 μIU/mL were retested with a second TSH assay. Discordant samples were evaluated on up to 8 FDA-approved TSH immunoassays and the TSHβ gene was sequenced. Retrospectively, thyroid function tests, diagnoses, and medications from 1.6 million individuals were analyzed.nnnRESULTSnOut of approximately 2 million individuals, we have identified a cohort of 20 hypothyroid and euthyroid patients of shared ethnicity with falsely undetectable TSH (<0.01 μIU/mL) in 4 of 8 commercially available TSH assays. Half of these individuals were initially treated based on repeated falsely undetectable TSH values (7 euthyroid patients were treated with methimazole and 2 hypothyroid patients had doses of levothyroxine decreased). In all cases, a retrospective chart review revealed that clinical assessments and free T4 and total T3 results were inconsistent with the undetectable TSH results. Specific antibodies failing to detect TSH in these cases were identified in the 4 affected assays. A novel TSHβ point mutation was identified.nnnCONCLUSIONSnOur data suggest that these individuals have a previously unrecognized, functionally normal, TSH variant to which some monoclonal antibodies fail to bind. To assure appropriate patient management, clinicians and laboratorians need to be aware that certain TSH variants may be undetectable in some hyperselective TSH assays.

Performance specifications of common chemistry analytes on the AU series of chemistry analyzers for miscellaneous body fluids

BACKGROUNDnBody fluids are frequently submitted to the clinical laboratory. Standard chemistry analyzers are generally not FDA approved for body fluid analysis and regulatory authorities require individual laboratories to validate their performance characteristics.nnnMETHODSnPrecision, linearity, accuracy, and sample stability were assessed for 10 different chemistry analytes using ascites, pleural, peritoneal dialysate, and synovial fluids on the Beckman Coulter (BC) AU5431. The effect of altered protein concentration and pH was investigated. Chemistry results from the AU5431 were compared with results from the BC AU5832 and the BC AU680. Additionally, 22 paired serum/pleural fluids were evaluated for their biochemical ability to distinguish a transudate from an exudate.nnnRESULTSnPrecision for each assay and body fluid type had a CV <14.5%. Recovery for the various analytes in all fluid types was 94.9-112%. CAP survey samples were within one SDI of the peer group. Sample pH, but not protein concentration, affected chemistry results. Inter-instrument comparison of results using the AU5431, AU5800, and AU680 was excellent. A combinatorial approach for discriminating between transudates and exudates had a sensitivity and specificity of 94% and 50%, respectively.nnnCONCLUSIONSnBody fluid samples with pH values within the acceptable range can be analyzed on the BC AU instruments.

Analytical sensitivity of four commonly used hCG point of care devices

BACKGROUNDnPoint of care (POC) hCG assays are often used to rule-out pregnancy and therefore diagnostic sensitivity, especially at low concentrations of hCG, is important. There are very few studies in the literature that seek to verify the claimed analytical sensitivity of hCG POC devices.nnnMETHODSnThe analytical sensitivity of four commonly used hCG POC devices (Alere hCG Combo Cassette, ICON 20 hCG, OSOM hCG Combo Test, and Sure-Vue Serum/Urine hCG-STAT) was challenged using urine samples (n=50) selected based on quantitative hCG concentrations. The majority of these specimens (n=40) had an hCG concentration between 20 and 200 U/L. Each specimen/device combination was reviewed by three individuals. Statistical calculations were performed using Stata 12.nnnRESULTSnThe analytical sensitivity of the OSOM was significantly lower inferior than that of the other POC devices. There was no significant difference in the sensitivity of the Alere, ICON 20 and Sure-Vue devices. There was no significant difference in the individual interpretation of the hCG POC results.nnnCONCLUSIONSnAll hCG POC devices evaluated in this study were susceptible to false negative results at low concentrations of urine hCG. Laboratorians and clinicians should be aware that there are limitations when using urine hCG POC devices to rule out early pregnancy.

Performance characteristics of the Beckman Coulter total βhCG (5th IS) assay

BACKGROUNDnBeckman Coulter recently released the first commercially available hCG reagent calibrated against the 5th International Standard (IS) for hCG (total βhCG (5th IS)). We performed a comprehensive analytical validation of this reagent.nnnMETHODSnPrecision experiments were completed using 3 concentrations of commercial quality control material. Linearity, sample stability, and analytical sensitivity were evaluated using pools of human serum. Reportable range was assessed by comparing manual dilutions to those performed by the instrument. Male and female reference intervals were established using residual serum specimens submitted for routine testing. Inter-lot variability of hCG assay reagent was assessed by analyzing serum specimens with detectable hCG using 2 different reagent lots. Inter-assay variability was established using 203 serum specimens analyzed for hCG on 6 different reagent platforms.nnnRESULTSnInter-day precision showed a CV of <6.0% for all concentrations of QC material. LOB, LOD, and LOQ were determined to be 0.3, 0.4, and 0.6 U/l, respectively. The Access (5th IS) reagent has an average positive bias of approximately 20% when compared to most platforms and the previous generation Beckman hCG assay. Results were consistent between lots. Female reference intervals varied by age: <1.0 U/l (<41 y), <6.0 U/l (41-50 y), and <8.0 U/l (>50 y). Male reference intervals were <2.0 U/l.nnnCONCLUSIONSnThe analytical performance of the total βhCG (5th IS) was established. Care should be taken to re-baseline patients needing serial monitoring and/or notify physicians when transitioning to the total βhCG (5th IS) reagent.