Preanalytical Factors Research Articles

Peripheral smear reckons the nature of IgM cold agglutinins: A case‐based study asserting the role of thermal amplitude and clonality

Peripheral smear reckons the nature of IgM cold agglutinins: A case‐based study asserting the role of thermal amplitude and clonality

Routine Next Generation Sequencing (NGS) Testing on Lung Adenocarcinoma Pap-stained Smears: Tumor Fraction and Cellularity as Preanalytical Factors Related with Success.

Routine Next Generation Sequencing (NGS) Testing on Lung Adenocarcinoma Pap-stained Smears: Tumor Fraction and Cellularity as Preanalytical Factors Related with Success.

Direct oral anticoagulants (DOACs): From the laboratory point of view.

Direct oral anticoagulants (DOACs) represent a new generation of drugs that have been increasingly used in the prevention and treatment of thromboembolic states. According to the mechanism of anticoagulant action, DOACs are divided into two groups: direct inhibitors of thrombin (dabigatran) and direct inhibitors of activated factor X (FXa) (rivaroxaban, apixaban, edoxaban, betrixaban). Compared to the vitamin K antagonists, DOACs are superior in terms of onset of action, pharmacokinetic and pharmacodynamics properties and fixed daily dose without the need for routine coagulation monitoring. Despite these advantages, there are clinical conditions in which laboratory measurement of DOACs should be performed. Although DOACs have an impact on screening haemostasis assays (prothrombin time, PT; activated partial thromboplastin time, aPTT; and thrombin time, TT), these tests are not appropriate for quantifying drug levels. Therefore, specific quantitative methods (LC-MS/MS as a gold standard method for all DOACs, coagulometric and chromogenic assays for dabigatran, and chromogenic anti-Xa assays with drug-specific calibrators for inhibitors of FXa) should only be used for determination of DOACs concentration. The aim of this review is to present all aspects of laboratory assessment of DOACs, including pre-analytical, analytical and post-analytical factors in the overall testing process with a special accent on the available specific quantitative methods for measurement of DOACs in circulation.

High false discovery rate of the Architect anti-HCV screening test in blood donors in Uganda and evaluation of an algorithm for confirmatory testing.

Adequate supplies of donor blood remain a major challenge in sub-Saharan Africa. This is exacerbated by a lack of confirmatory testing for transfusion-transmitted infections by blood transfusion services (BTS), leading to significant blood disposal owing to putatively high seroprevalence rates amongst Ugandan blood donors. We aimed to ascertain the false discovery rate of the Architect anti-hepatitis C virus (HCV) screening assay and categorize screen-reactive samples into three groups: presumed false positive, active and past infection, and develop an algorithm for confirmatory testing. A total of 470 screen-reactive HCV blood donations were retested using the Architect anti-HCV assay, an alternative antibody test (SD Biosensor) and a core antigen (cAg) test. signal-to cut-off (S/CO) ratios and pre-analytical factors (centrifugation speed, haemolysis check, time between collection and testing) were recorded. Based on the S/CO ratio evaluation, we propose a testing algorithm to guide supplemental tests. The false discovery rate of the Architect anti-HCV assay was 0.84 as 395/470 (84%) screen-reactive samples had no evidence of HCV infection (SD Biosensor and cAg negative) (presumed false positive), 38/470 (8.1%) were antigenaemic, and 32/470 (6.8%) had evidence of past infection. The median S/CO ratios of the presumed false-positive and active infection samples were 1.8 and 17.3, respectively. The positive predictive value of HCV positivity in samples with ratios above 12 was 91.8%. On retesting, 104/470 (22.1%) samples became negative. The Architect anti-HCV assay has a very high false discovery rate in Ugandan BTSs, leading to excessive blood disposal. Pre-analytical factors likely contribute to this. An introduction of confirmatory testing using an algorithm based on S/CO ratio evaluation could limit unnecessary blood wastage and donor deferral.

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study

AbstractKi67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

Key methodological challenges in detecting circulating miRNAs in different biofluids.

The technological advancement in diagnostic techniques has immensely improved the capability of predicting disease progression. Yet, there is a great interest in developing newer biomarkers that can enhance disease risk prediction thereby minimising the associated morbidity and mortality. Circulating miRNAs, a non-coding RNA molecule, are critical regulators in the pathophysiology of various complex multifactorial diseases. In recent years, circulating miRNAs have been enormously studied and are considered as an emerging biomarker due to their easy accessibility, stability, and detection by sequence-specific amplification methods. However, there is a distinct lack of consensus regarding the preanalytical factors such as preferred sample selection, methodological aspects, etc that may independently or together influence the detection of circulating miRNAs resulting in erroneous expression profiles. Therefore, the present review makes an attempt to highlight the various pre-analytical and analytical factors that can potentially influence the circulating miRNA levels. Literature on circulating miRNA's stability, processing and quantitation in different biofluids along with the effect of various controllable and uncontrollable factors influencing circulating miRNA expression have been summarised in the current review.

Stability of Selected Biochemical Analytes in Serum Stored at Room Temperature for 24 Hours at Kericho County Referral Hospital, Kenya

Accurate and reliable medical laboratory test results are essential for correct patient diagnosis and management. This is however influenced by many preanalytical, analytical, and post-analytical factors. A general problem faced by most clinical laboratories is the inability to timely process all blood samples received due to overwhelming workload, reagents stock-outs, and equipment breakdown, necessitating storage which predisposes analytes to deterioration. To ensure accurate results from stored samples, the determination of the stability of the analytes under various environmental conditions and storage periods is important in informing acceptable sample handling and storage. This study was designed to experimentally observe changes and evaluate the stability of eleven biochemical analytes in serum samples from 20 randomly sampled healthy volunteers stored at room temperature over 24 hours at Kericho County Referral Hospital laboratory. 10mls of venous blood was drawn from each volunteer, serum was separated and five aliquot sets were prepared. Analysis was done on a set immediately, at 2, 4, 8, and 24 hours, to determine the concentration of the selected analytes on Human Star 100 chemistry analyser. Student pair T-test and Wilcoxon rank were used to analyse the data. Chloride, Creatinine, Glucose, Potassium, Sodium, Urea, Alkaline Phosphatase, Alanine Amino Transferase and Aspartate Amino Transferase registered stability up to 24 hours, with mean percentage differences less the respective calculated reference change value of 4.54, 35.97, 13.14, 10.89, 2.16, 32.15, 16.48, 42.25 and 42.52. Direct and total bilirubin showed stability only up to 2 and 4 hours, respectively, with mean percentage differences of -2.6 and 1.07 against respective calculated reference change values of 1.14 and 0.675 in the subsequent analysis. This study recommends immediate analysis of serum for total and direct bilirubin, while electrolytes, enzymatic analytes and other metabolic waste and substrates can be analysed within 24 hours without extra storage precautions. If testing is delayed, then effective storage including a dark room, refrigeration, or freezing, should be applied to maintain analytes stability for reliable results.

Serum GFAP- reference interval and preanalytical properties in Danish adults.

Glial fibrillary acidic protein (GFAP) is a promising biomarker that could potentially contribute to diagnosis and prognosis in neurological diseases. The biomarker is approaching clinical use but the reference interval for serum GFAP remains to be established, and knowledge about the effect of preanalytical factors is also limited. Serum samples from 371 apparently healthy reference subjects, 21-90 years of age, were measured by a single-molecule array (Simoa) assay. Continuous reference intervals were modelled using non-parametric quantile regression and compared with traditional age-partitioned non-parametric reference intervals established according to the Clinical and Laboratory Standards Institute (CLSI) guideline C28-A3. The following preanalytical conditions were also examined: stability in whole blood at room temperature (RT), stability in serum at RT and-20°C, repeated freeze-thaw cycles, and haemolysis. The continuous reference interval showed good overall agreement with the traditional age-partitioned reference intervals of 25-136ng/L, 34-242ng/L, and 5-438ng/L for the age groups 20-39, 40-64, and 65-90 years, respectively. Both types of reference intervals showed increasing levels and variability of serum GFAP with age. In the preanalytical tests, the mean changes from baseline were 2.3% (95% CI:-2.4%, 6.9%) in whole blood after 9h at RT, 3.1% (95% CI:-4.5%, 10.7%) in serum after 7days at RT, 10.4% (95% CI:-6.0%, 26.8%) in serum after 133days at-20°C, and 10.4% (95% CI: 9.5%, 11.4%) after three freeze-thaw cycles. The study establishes age-dependent reference ranges for serum GFAP in adults and demonstrates overall good stability of the biomarker.

The Impact of the Anticoagulant Type in Blood Collection Tubes on Circulating Extracellular Plasma MicroRNA Profiles Revealed by Small RNA Sequencing.

Pre-analytical factors have a significant influence on circulating microRNA (miRNA) profiling. The aim of this study was a comprehensive assessment of the impact of the anticoagulant type in blood collection tubes on circulating plasma miRNA profiles using small RNA sequencing. Blood from ten healthy participants (five males and five females from 25 to 40 years old) was taken in collection tubes with four different anticoagulants: acid citrate dextrose (ACD-B), sodium citrate, citrate-theophylline-adenosine-dipyridamole (CTAD) and dipotassium-ethylenediaminetetraacetic acid (K2 EDTA). Platelet-free plasma samples were obtained by double centrifugation. EDTA plasma samples had elevated levels of hemolysis compared to samples obtained using other anticoagulants. Small RNA was extracted from plasma samples and small RNA sequencing was performed on the Illumina NextSeq 500 system. A total of 30 samples had been successfully sequenced starting from ~1 M reads mapped to miRNAs, allowing us to analyze their diversity and isoform content. The principal component analysis showed that the EDTA samples have distinct circulating plasma miRNA profiles compared to samples obtained using other anticoagulants. We selected 50 miRNA species that were differentially expressed between the sample groups based on the type of anticoagulant. We found that the EDTA samples had elevated levels of miRNAs which are abundant in red blood cells (RBC) and associated with hemolysis, while the levels of some platelet-specific miRNAs in these samples were lowered. The ratio between RBC-derived and platelet-derived miRNAs differed between the EDTA samples and other sample groups, which was validated by quantitative PCR. This study provides full plasma miRNA profiles of 10 healthy adults, compares them with previous studies and shows that the profile of circulating miRNAs in the EDTA plasma samples is altered primarily due to an increased level of hemolysis.

External Quality Assurance programs for processing methods provide evidence on impact of preanalytical variables

An annual External Quality Assurance (EQA) program has been provided to processing laboratories over the last ten years, allowing them to assess the performance of their processing methods, such as nucleic acid extractions or peripheral blood mononuclear cell (PBMC) isolation and cryopreservation. The objective of this study was to perform a global analysis on almost 1000 EQA scheme/participant data in order to assess (i) the impact of critical preanalytical factors on quantitative or qualitative attributes of different types of specimens and (ii) laboratory performance pattern over time. Statistical analysis was performed within each EQA scheme based on categorized preanalytical data provided by the participants and on centralized measurements of relevant quality attributes of the produced specimens (z-scores): DNA, cell-free (cf)DNA or RNA extraction from blood, DNA or RNA extraction from formalin fixed tissue, DNA or RNA extraction from frozen tissue, DNA extraction from saliva or stool, viable PBMC isolation and cryopreservation. The most critical preanalytical factors in nucleic acid extraction schemes were the nucleic acid extraction method and kit, the elution buffer, the enzymes used during extraction, the input material quantity and the storage temperature. Several indications of laboratory performance improvement over time could be seen. The conclusions are that EQA for processing methods provides unique evidence-based insights into the impact of preanalytical factors and the comparative performance of different processing methods and kits, while supporting laboratories in validating their processing methods.

Saliva-based SARS-CoV-2 serology using at-home collection kits returned via mail

Serology provides tools for epidemiologic studies, and may have a role in vaccine prioritization and selection. Automated serologic testing of saliva, especially specimens that are self-collected at home and sent to a laboratory via the mail without refrigeration, could be a highly-scalable strategy for population-wide testing. In this prospective study, non-vaccinated patients were recruited after PCR testing to self-collect saliva and return their specimens via mail. Longitudinal specimens were analyzed in order to monitor seroconversion in the weeks after a diagnostic PCR test for SARS-CoV-2. Diverse users self-collected saliva and returned specimens via mail in compliance with shipping regulations. At our pre-established threshold (0.963 AU/mL), salivary IgG reactivity to full-length spike protein achieved 95.8% sensitivity and 92.4% specificity at 2–4 weeks after diagnostic testing, which is comparable to the typical sensitivity and specificity achieved for serum testing. Reactivity to N antigen also was detected with 92.6% sensitivity and 90.7% specificity at 4–8 weeks after diagnostic testing. Moreover, serologic testing for endemic coronaviruses performed in multiplex with SARS-CoV-2 antigens has the potential to identify samples that may require retesting due to effects of pre-analytical factors. The easy-to-use saliva collection kit, coupled with thresholds for positivity and methods of flagging samples for retest, provides a framework for large-scale serosurveillance of SARS-CoV-2.

Comparison of Jugular vs. Saphenous Blood Samples, Intrarater and In-Between Device Reliability of Clinically Used ROTEM S Parameters in Dogs.

Simple SummaryViscoelastic coagulation tests such as rotational Thromboelastometry (ROTEM) have many theoretical advantages compared to traditional coagulation testing. As a point-of-care diagnostic device, ROTEM results are directly part of treatment decisions. Therefore, it is crucial to know its reliability and precision. Most recommendations for ROTEM S analyses originate from Thromboelastography (TEG), another viscoelastic coagulation assay. However, evidence about how preanalytical and analytical factors, such as sample collection technique, sample handling and the analysis itself, influence ROTEM results is scarce. Due to the absence of a gold standard method, we assessed accuracy with the coefficient of variation and intraclass correlation coefficient and examined the influence of blood collection site, as well as intrarater and in-between device variability, on ROTEM S results of clinically healthy dogs. We found significant changes between ROTEM S parameters from different blood collection sites and significant intrarater and in-between device variability. These findings were most prominent in tissue-factor-activated tests. To ensure patient safety, we therefore suggest running duplicate measurements and to interpret results obtained from tissue-factor-activated tests with caution, since some of their coefficients of variation were moderate to high.Rotational Thromboelastometry (ROTEM) allows for the global assessment of hemostasis in whole blood samples. Preanalytical and analytical factors may influence test results, and data about the reliability and reproducibility of lyophilized ROTEM tests are scarce. Therefore, the objective of this study was to evaluate the influence of blood collection site on ROTEM S parameters and to assess intrarater and in-between device variability. A total of thirty, healthy, staff-owned dogs were included. Blood collection and ROTEM analysis were performed by trained staff according to a standardized protocol. Extrinsically activated (tissue factor; Ex-TEM S), with the addition of cytochalasin for platelet inhibition (Fib-TEM S), and intrinsically activated (In-TEM) analyses were performed. Analysis of our data showed significant variability for various Ex-TEM S and Fib-TEM S parameters from different collection sites and intrarater and in-between device measurements. We conclude that serial monitoring with ROTEM should be performed on the same device, with blood always taken from the same collection site using a standardized blood sampling technique. While In-TEM S, apart from maximum lysis, showed very stable and reliable results, we suggest interpreting especially clotting and clot formation parameters from Ex-TEM S and Fib-TEM S tests with caution and using duplicate measurements to detect outliers and to prevent initiation of incorrect therapies.

Influence of Trimethylamine N-Oxide on Platelet Activation.

Microbiome-derived trimethylamine N-oxide (TMAO) has been associated with platelet hyperreactivity and subsequent atherogenesis. Whether physiological TMAO-levels influence platelet-derived lipid mediators remains unknown. Little is known about pre-analytic factors potentially influencing TMAO concentrations. We aimed at developing a quantitative LC-MS/MS method to investigate in-vivo and in-vitro pre-analytical factors in TMAO analysis to properly assess the proposed activating effect of TMAO on platelets. TMAO, betaine, carnitine, and choline were analyzed by HILIC-ESI-MS/MS within 6 min total run time. Method validation included investigation of reproducibility, recovery, sensitivity, and in-vitro pre-analytical factors. A 24-h monitoring experiment was performed, evaluating in-vivo pre-analytical factors like daytime or diet. Finally, the effects of different TMAO concentrations on platelet activation and corresponding alterations of platelet-derived eicosanoid release were analyzed. The method showed high reproducibility (CVs ≤ 5.3%), good recovery rates (96–98%), and negligible in-vitro pre-analytical effects. The influence of in-vivo pre-analytical factors on TMAO levels was not observable within the applied experimental conditions. We did not find any correlation between TMAO levels and platelet activation at physiological TMAO concentrations, whereas platelet-derived eicosanoids presented activation of the cyclooxygenase and lipoxygenase pathways. In contrast to previously published results, we did not find any indications regarding diet dependency or circadian rhythmicity of TMAO levels. Our results do not support the hypothesis that TMAO increases platelet responsiveness via the release of lipid-mediators.

Broad application prospects of bone turnover markers in pediatrics.

BackgroundBone turnover markers (BTMs) have been studied for application in clinical medicine. However, BTMs in children are challenging, and few studies explore these BTMs in children. The application of BTMs is complicated mainly due to pre‐analytical factors, variable reference intervals of age‐ and sex‐related BTMs for adolescents and children in different regions and laboratories. Therefore, laboratory testing of BTMs is critical for understanding pediatric bone development and metabolism, which provides additional information about bone development and diseases.MethodsLiterature search was conducted using the MeSH term “child” combined with the terms that bone turnover markers such as “osteocalcin,” “Procollagen type I N‐terminal propeptide,” “procollagen type I C‐terminal propeptide,” “osteocalcin,” “N‐terminal cross‐linked telopeptide,” and “C‐terminal cross‐linked telopeptide,” Several databases including Web of Science, Google Scholar, and PubMed were searched to obtain the relevant studies.ResultsBTMs represent the combined effects of skeletal development, growth, and remodeling in children, which can be used in clinical pediatrics to assist in the diagnosis and prognosis of bone metabolic disorders.ConclusionBTMs are clearly helpful for diagnosis and monitoring of bone growth and development as well as bone metabolic disorders.

Methodological considerations for circulating long noncoding RNA quantification.

In the past decade, significant resources have been invested in long noncoding RNA (lncRNA) research. Despite the knowledge available, we are far from incorporation of lncRNA into clinical practice. Here, we emphasize the technical challenges in the field, hoping to provoke a response leading to new consensus and guidelines.

Preanalytical Pitfalls in Untargeted Plasma Nuclear Magnetic Resonance Metabolomics of Endocrine Hypertension.

Despite considerable morbidity and mortality, numerous cases of endocrine hypertension (EHT) forms, including primary aldosteronism (PA), pheochromocytoma and functional paraganglioma (PPGL), and Cushing’s syndrome (CS), remain undetected. We aimed to establish signatures for the different forms of EHT, investigate potentially confounding effects and establish unbiased disease biomarkers. Plasma samples were obtained from 13 biobanks across seven countries and analyzed using untargeted NMR metabolomics. We compared unstratified samples of 106 PHT patients to 231 EHT patients, including 104 PA, 94 PPGL and 33 CS patients. Spectra were subjected to a multivariate statistical comparison of PHT to EHT forms and the associated signatures were obtained. Three approaches were applied to investigate and correct confounding effects. Though we found signatures that could separate PHT from EHT forms, there were also key similarities with the signatures of sample center of origin and sample age. The study design restricted the applicability of the corrections employed. With the samples that were available, no biomarkers for PHT vs. EHT could be identified. The complexity of the confounding effects, evidenced by their robustness to correction approaches, highlighted the need for a consensus on how to deal with variabilities probably attributed to preanalytical factors in retrospective, multicenter metabolomics studies.

Plasma calprotectin – preanalytical stability and interference from hemolysis

As an activation product of neutrophil granulocytes, calprotectin has been widely used in fecal samples for diagnosis and monitoring of patients with inflammatory bowel disease. However, fecal sample collection is cumbersome, and pre-analytical sources of error are plentiful. Therefore, plasma calprotectin is being investigated as a promising new biomarker. To utilize any biomarker, pre-analytical factors such as stability and susceptibility to interference from hemolysis must be established. We present precision estimates, stability results as well as interference study on plasma calprotectin in EDTA-plasma using the Thermo Fischer Phadia 250 EliATM Calprotectin immunoassay. Precision was estimated by the use of patient pools as well as internal quality controls provided by the manufacturer. Coefficients of variance were below 6.9% for patient samples. Calprotectin was stable in EDTA plasma after storage at 5–8 °C for up to 4 days, as well as after long-term storage at −20 °C. Susceptibility to interference from hemolysis was high, especially for low concentrations of calprotectin (<50 ng/mL) where hemoglobin levels above 0.02 mmol/L lead to false increase in calprotectin concentrations of up to 100%.

A systematic review and meta-analysis of preanalytical factors and methodological differences influencing the measurement of circulating vascular endothelial growth factor.

BackgroundIntraocular treatment with antibodies targeting vascular endothelial growth factor (anti-VEGF) inhibits pathological vessel growth in adults and preterm infants. Recently, concerns regarding the impact of anti-VEGF treatment on systemic VEGF levels in preterm infants have been raised. Earlier studies suggest that preanalytical and methodological parameters impact analytical VEGF concentrations, but we have not found a comprehensive systematic review covering preanalytical procedures and methods for VEGF measurements.ObjectiveThis review aimed to evaluate the most critical factors during sample collection, sample handling, and the analytical methods that influence VEGF levels and therefore should be considered when planning a prospective collection of samples to get reproducible, comparable results.Material and methodsPubMed and Scopus databases were searched 2021/Nov/11. In addition, identification of records via other methods included reference, citation, and Google Scholar searches. Rayyan QCRI was used to handle duplicates and the selection process. Publications reporting preanalytical handling and/or methodological comparisons using human blood samples were included. Exclusion criteria were biological, environmental, genetic, or physiological factors affecting VEGF. The data extraction sheets included bias assessment using the QUADAS-2 tool, evaluating patient selection, index-test, reference standard, and flow and timing. Concentrations of VEGF and results from statistical comparisons of analytical methods and/or preanalytical sample handling and/or different sample systems were extracted. The publications covering preanalytical procedures were further categorized based on the stage of the preanalytical procedure. Meta-analysis was used to visualize VEGF concentrations among healthy individuals. The quality of evidence was rated according to GRADE.ResultsWe identified 1596 publications, and, after the screening process, 43 were considered eligible for this systematic review. The risk of bias estimation was difficult for 2/4 domains due to non-reported information. Four critical steps in the preanalytical process that impacted VEGF quantification were identified: blood drawing and the handling before, during, and after centrifugation. Sub-categorization of those elements resulted in nine findings, rated from moderate to very low evidence grade. The choice of sample system was the most reported factor. VEGF levels (mean [95% CI]) in serum (n = 906, 20 publications), (252.5 [213.1–291.9] pg/mL), were approximated to ninefold higher than in plasma (n = 1122, 23 publications), (27.8 [23.6–32.1] pg/mL), based on summarized VEGF levels with meta-analysis. Notably, most reported plasma levels were below the calibration range of the used method.ConclusionWhen measuring circulating VEGF levels, choice of sample system and sample handling are important factors to consider for ensuring high reproducibility and allowing study comparisons. Protocol: CRD42020192433

High Incidence of Partial Biotinidase Deficiency in the First 3 Years of a Regional Newborn Screening Program in Italy.

Biotinidase deficiency (BD) is an autosomal recessive inherited disorder in which the enzyme biotinidase is totally or partially defective and the vitamin biotin is not recycled. BD meets the major criteria for a population screening program. Newborn bloodspot screening (NBS) allows early diagnosis of BD, thus preventing the high morbidity and mortality associated with untreated disease. Both profound and partial BD variant can be detected by NBS test, and serum enzyme activity and/or mutational analysis are required for definitive diagnosis. In Italy, BD is included in the screening panel for inborn errors of metabolism (IEMs) that has been declared mandatory in 2016. We analyzed the data of the first 3 years of the NBS for BD in our region (Abruzzo, Italy), with the aim to describe the outcomes of this recently introduced screening program. In over 26,393 newborns screened, we found 2 carriers and 16 cases with genotype associated with partial BD. Since the serum biotinidase assay has been recently introduced in our algorithm, only three of our newborns met the criteria of genetic and biochemical confirmation, with an incidence of 1:8797, which is in the high range of what has been reported in the literature. All affected infants carried the 1330G>C (D444H) variant in compound heterozygosis, with variants known to be associated with profound BD. A variant previously not described and likely pathogenic was found in one newborn. None of the infants had signs or symptoms. The study of the distribution of the enzyme activity in our population allowed us to validate the adopted cutoff with which the program has a positive predictive value of 18% and to analyze some preanalytical factors influencing biotinidase activity: A correlation of the enzyme activity with gestational age and time at specimen collection was found. Lower mean values of enzyme activity were found in infants born in the summer.

Protein Profiling of Breast Cancer for Treatment Decision-Making.

The increasing use of neoadjuvant therapy has resulted in therapeutic decisions being made on the basis of diagnostic needle core biopsy. For many patients, this method might yield the only fragment of tumor available for biomarker analysis, necessitating judicious use. Many multiplex protein analytic methods have been developed that employ fluorescence or other tags to overcome the limitations of immunohistochemistry while still retaining the spatial annotation. Interpretation of the data can be difficult because of the limitations of the human eye. Computational deconvolution of the signals may be necessary for some of these methods to enable identification of cell-specific localization and coexpression of biomarkers. Herein, we present the different methods that are coming of age and their application in cancer research, with a focus on breast cancer. We also discuss the limitations, which include high costs and long turnaround times. The methods are also based on the premise that preanalytical factors will have identical impact on all proteins analyzed. There is a need to establish standards to normalize the data and enable cross-sample comparisons. In spite of these limitations, the multiplex technologies are extremely valuable discovery tools and can provide novel insights into the biology of cancer and mechanisms of drug resistance.