Jessica M. Colón-Franco

Dynamic remodeling of the actin cytoskeleton by FMNL1γ is required for structural maintenance of the Golgi complex.

Formin-like 1 (FMNL1) is a member of the formin family of actin nucleators, and is one of the few formins for which in vitro activities have been well characterized. However, the functional roles of this mammalian formin remain ill-defined. In particular, it is unclear how the unique in vitro biochemical properties of FMNL1 relate to its regulation of cellular processes. Here, we demonstrate that FMNL1 depletion caused a dramatic increase in cellular F-actin content, which resulted in Golgi complex fragmentation. Moreover, increased F-actin and maintenance of Golgi structure were distinctly regulated by the gamma isoform of FMNL1, which required binding to actin. Importantly, in addition to Golgi fragmentation, increased F-actin content in the absence of FMNL1 also led to cation-independent mannose 6-phosphate receptor dispersal, lysosomal enlargement and missorting of cathepsin D. Taken together, our data support a model in which FMNL1 regulates cellular F-actin levels required to maintain structural integrity of the Golgi complex and lysosomes.

Validation of a hand-held point of care device for lactate in adult and pediatric patients using traditional and locally-smoothed median and maximum absolute difference curves

BACKGROUND Lactate is commonly used in septic patients and is a viable biomarker for trauma patients. Its pre-hospital use could assist triaging and managing patients with these conditions. METHODS We evaluated the analytical performance of the point-of-care (POC) StatStrip Xpress Lactate Meter (Nova Biomedical) and compared it to the ABL 800 (Radiometer). We measured lactate in 250 adult and 250 pediatric whole blood samples in 2 laboratories. The performance of the POC meter was assessed by traditional linear regression and Bland-Altman plots, and locally-smoothed (LS) median absolute difference and maximum absolute difference (MAD and MaxAD) curves. RESULTS The StatStrip was linear with acceptable reproducibility at clinically relevant concentrations. Correlation with the ABL800 showed a negative bias for both populations with slope, bias ±SD (% bias) of 0.78, -0.4±0.7 (-14.5%) in children and 0.80-0.3±0.6 (-13.3%) in adults. The proportional bias appeared more significant at concentrations >4mmol/l (36.0mg/dl). The StatStrip misclassified 7.6 and 8.8% pediatric and adult samples, respectively, to lower risk categories defined using guidelines driven cut-offs. The LS MAD curves identified one breakout, concentration where the LS MAD exceeds the total allowable error limit of 0.3mmol/l (2.7mg/dl), at lactate concentrations of 3.8 and 3.2mmol/l (34.2 and 28.8mg/dl) in the pediatric and adult curves, respectively. Breakthroughs, points at which the LS MaxAD curve exceeds the 95th percentile of MaxADs, occur at concentrations above 7.5mmol/l (67.6mg/dl) for both populations where the performance of the POC meter became erratic. We concluded that if serial lactate measurements are performed, the same method should be used for baseline and follow up measurements. The LS MAD and LS MaxAD curves allowed visual and quantitative mapping of the performance of the lactate POC meter over the range of concentrations measured. CONCLUSIONS This approach seems useful for the identification of points at which the performance of a POC meter differs significantly from a comparison method and thresholds of poor analytical performance.

Resolution of an Apparent Hook Effect in Roche Partner DRI Oxycodone Immunoassay

A presumed hook effect in the semiquantitative DRI Oxycodone immunoassay, OXY3S (Cobas Integra, Roche Diagnostics), was investigated in 14 urine samples with gas chromatography/mass spectrometry (GC-MS) >10,000 ng/mL but OXY3S <1,000 ng/mL. These samples included the index case, a false-negative OXY3S result with >75,000 ng/mL oxycodone + oxymorphone by GC-MS confirmation. Patient samples needed 2- to 16-fold dilution to obtain the correct OXY3S response. The OXY3S test did not hook at high-spiked concentrations of oxycodone, oxymorphone or oxymorphone-3β-d-glucuronide in drug-free urine. The OXY3S test parameters were replicated in a development channel on the Cobas using DRI Reagents (Microgenics, CA, USA) and were subsequently modified. Delayed sample addition or doubling of Reagent 1 (R1: antibody/substrate/co-factor) yielded maximal immunoassay response (>10,000 ng/mL) in 12 of 14 and 14 of 14 undiluted patient samples, respectively. Supplementation of R1 with substrate alone did not correctly recover oxycodone from any of the samples, while co-factor supplementation resulted a maximal OXY3S response in 13 of 14 samples. The remaining (index) sample could only be corrected by supplemental R1. The semiquantitative utility of the DRI Oxycodone assay is questionable. Although the precise cause of the under-recovery could not be determined, the modification presented permits reliable oxycodone determination at the high concentrations frequently seen in clinical urine samples.

Current and Emerging Multianalyte Assays with Algorithmic Analyses—Are Laboratories Ready for Clinical Adoption?

Over the past decade, there have been a rising number of clinically used tests that combine 2 or more biochemical or molecular assays, demographics, and clinical information into an algorithm to generate diagnostic, prognostic, or predictive information for a specific disease. The concept of multianalyte analyses is relatively new in the field of laboratory medicine. Dating back to the 1980s, prenatal screening for fetal abnormalities, such as Down syndrome, by use of maternal biomarkers is among the pioneer tests that use algorithmic analyses for risk assessment. Yet, the number of multianalyte algorithms used clinically remains modest. The American Medical Association provides current procedural terminology (CPT)8 codes for 20 multianalyte assays with algorithmic analyses (MAAAs.) Among these, 9 consist of biochemical markers detected by immunoassay or mass spectrometry, with or without other clinical information; 11 use molecular genetics markers; and 1 is for generic use. In this Q&A, we refer to multivariable tests with risk scores as MAAAs, although not all of them have an associated MAAA CPT code. Generally speaking, MAAAs aim at improving diagnostics for diseases in which single biomarkers have limited clinical validity. The Prostate Health Index (Beckman Coulter) and the 4Kscore® (GenPath) exemplify some of these strategies for prostate cancer detection. Likewise, multianalyte analyses such as the Risk of Ovarian Malignancy Algorithm (ROMA®) and OVA1® and its second-generation OVERATM (Vermillion Inc.) improve upon the suboptimal performance of the tumor marker CA125 in the differential diagnosis and likelihood of ovarian carcinoma in women presenting with a pelvic mass. While both have Food and Drug Administration (FDA) clearance, ROMA is a nonproprietary algorithm cleared on various commercial immunoassays (Fujirebio Diagnostics, Inc.; Abbott Laboratories; Roche Diagnostics). Early identification of acute kidney injury is another area in which an FDA-approved multianalyte test, Nephrocheck® (Astute Medical), …

Unexpected Change in Thyroglobulin Concentration

A high thyroglobulin (Tg) result was questioned as a possible laboratory error (Table 1). The sample corresponded to a 66-year-old male with a history of recurrent stage IV papillary thyroid carcinoma status post-thyroidectomy, routinely monitored for recurrence using Tg results and imaging studies. Suspicious findings on neck ultrasonography were followed-up by fine-needle aspiration (FNA)2 of a cervical lymph node. Cytology FNA results showed cancerous thyroid …

A Diabetic Newborn

A sample submitted for hemoglobin (Hb) evaluation on a 5-day-old premature male infant with intestinal perforation, intraventricular hemorrhage, anemia, and sepsis contained 61.1% Hb A, 23.7% Hb F, 2.1% Hb A2, and peaks of 8.2% and 5.2% in the P2 and P3 regions of the Bio-Rad Variant II HPLC β thalassemia assay (Fig. 1). Isoelectric focusing …