Introduction:To ensure the quality of the new-born screening (NBS), our laboratory reviewed the analytical procedure to detect subjective steps that may represent a risk to the patient. Two subjective activities were identified in the extra-analytical phases: the classification of dried blood spots (DBS) according to their quality and the assignment of haemoglobin patterns. To keep these activities under control, inter-rater studies were implemented. This study aimed to evaluate the inter-rater reliability and the effectiveness of the measures taken to improve the agreement between observers, to assure NBS results' quality.Materials and methods:Dried blood spots specimens were used for the inter-rater studies. Ten studies were performed to assess DBS quality classification, and four to assess the assignment of haemoglobin patterns. Krippendorff's alpha test was used to estimate inter-rater reliability. Causes were investigated when alpha values were below 0.80.Results:For both activities, the reliability obtained in the first studies was inadequate. After investigation, we detected that the criterion to classify a DBS as scant was not consolidated, and also a lack of consensus on whether or not to report Bart's haemoglobin depending on its percentage. Alpha estimates became higher once the training was reinforced and a consensus about the appropriate criteria to be applied was reached.Conclusion:Inter-rater reliability assessment helped us to ensure the quality of subjective activities that could add variability to NBS results. Furthermore, the evolution of the alpha value over time allowed us to verify the effectiveness of the measures adopted.

In the field of laboratory medicine, proficiency testing is a vehicle used to improve the reliability of reported results. When proficiency tests are unavailable for a given analyte, an alternative approach is required to ensure adherence to the International Organization for Standardization (ISO) 15189:2012 standard. In this study, we report the results of a split-sample testing program performed as an alternative to a formal PT. This testing method was based on recommendations provided in the Clinical and Laboratory Standards Institute (CLSI) QMS24 guideline. Two different laboratories measured, in duplicate, the heparan sulfate concentration in five samples using ultra-performance liquid chromatography and tandem mass spectrometry. The data analysis to determine the criterion used for the comparability assessment between the two laboratories was based on Appendix E of the QMS24 guideline. Mean interlaboratory differences fell within the maximum allowable differences calculated from the application of the QMS24 guideline, indicating that the results obtained by the two laboratories were comparable across the concentrations tested. Application of the QMS24 split-sample testing procedure allows laboratories to objectively assess test results, thus providing the evidence needed to face an accreditation audit with confidence. However, due to the limitations of statistical analyses in small samples (participants and/or materials), laboratory specialists should assess whether the maximum allowable differences obtained are suitable for the intended use, and make adjustments if necessary.

Objective: To explore the use of a new molecular work-up based on the stepwise use of Quantitative Fluorescence PCR (QF-PCR) extended to eight chromosomes and single nucleotide polymorphism array (SNP-array) in chorionic villi obtained by chorionic villi sampling (CVS) offered to women experiencing an early pregnancy loss. Methods: During a 3-year period (January 2016–December 2018), CVS was offered to women experiencing an early pregnancy loss before the evacuation of the products of conception (POC) to retrieve chorionic villi, irrespective of the number of previous losses. A new molecular work-up was prospectively assayed encompassing a first QF-PCR round (with the 21, 18, 13, 7, X, and Y chromosomes), a second QF-PCR round (with the 15, 16, and 22 chromosomes), and a high resolution SNP-array in those cases with normal QF-PCR results. A control group in which POC were collected after surgical uterine evacuation was used to be compared with the intervention group. Results: Around 459 women were enrolled in the intervention group (CVS) and 185 in the control group (POC after uterine evacuation). The QF-PCR testing success rates were significantly higher in the intervention group (98.5%: 452/459) as compared to the control group (74%: 109/147; p < 0.001), while the chromosomal anomaly rate at the two QF-PCR rounds was similar between the two groups: 52% (234/452) in the intervention and 42% (46/109) in the control group ( p = 0.073). The SNP-array was performed in 202 QF-PCR normal samples of the intervention group and revealed 67 (33%) atypical chromosomal anomalies (>10 Mb), 5 (2.5%) submicroscopic pathogenic copy number variants, and 2 (1%) variant of uncertain significance (VOUS). Conclusion: Eighty-two percent of women experiencing an early pregnancy loss opted for a CVS. The testing success rates were higher in the intervention group (CVS; 98%) as compared to the control group (POC; 74%). The overall yields were 52% by QF-PCR (including three complete hydatiform moles), and 16% by SNP-array, including 15% atypical chromosomal anomalies and 1.1% submicroscopic pathogenic copy number variants.

INTRODUCTION:Laboratories minimize risks through quality control but analytical errors still occur. Risk management can improve the quality of processes and increase patient safety. This study aims to use the failure mode and effect analysis (FMEA) to assess the analytical performance and measure the effectiveness of the risk mitigation actions implemented.MATERIALS AND METHODS:The measurands to be included in the study were selected based on the measurement errors obtained by participating in an External Quality Assessment (EQA) Scheme. These EQA results were used to perform an FMEA of the year 2017, providing a risk priority number that was converted into a Sigma value (σFMEA). A root-cause analysis was done when σFMEA was lower than 3. Once the causes were determined, corrective measures were implemented. An FMEA of 2018 was carried out to verify the effectiveness of the actions taken.RESULTS:The FMEA of 2017 showed that alkaline phosphatase (ALP) and sodium (Na) presented a σFMEA of less than 3. The FMEA of 2018 revealed that none of the measurands presented a σFMEA below 3 and that σFMEA for ALP and Na had increased.CONCLUSIONS:Failure mode and effect analysis is a useful tool to assess the analytical performance, solve problems and evaluate the effectiveness of the actions taken. Moreover, the proposed methodology allows to standardize the scoring of the scales, as well as the evaluation and prioritization of risks.

Abstract STUDY QUESTION Can maternal plasma cell-free DNA (cfDNA) detect chromosomal anomalies in early pregnancy loss (EPL) and recurrent pregnancy loss (RPL)? SUMMARY ANSWER Genome-wide cfDNA testing can serve as an alternative to cytogenetic analysis in products of conception (POCs) in RPLs and can guide further management. WHAT IS KNOWN ALREADY Random chromosomal anomalies are the single most common cause for EPL and RPL. Cytogenetic analysis in POCs may be used to direct management in RPL because the detection of random chromosomal anomalies can eliminate further unwarranted testing. STUDY DESIGN, SIZE, DURATION This was a prospective diagnostic test study from March 2018 to January 2019 of 109 patients experiencing pregnancy loss before 14 weeks gestation at a tertiary-care academic medical center. PARTICIPANTS/MATERIALS, SETTING, METHODS Blood samples were drawn for genome-wide cfDNA testing prior to chorionic villous sampling for cytogenetic analysis of POCs with both short-term cultures (STCs) and long-term cultures (LTCs). Final analysis included 86 patients with non-mosaic cytogenetic results in POCs and available cfDNA results. Aneuploidy detection rates by cfDNA testing and POC cytogenetic analysis were compared. The first 50 samples served as the Training Set to establish pregnancy loss-specific log-likelihood ratio (LLR) thresholds using receiver-operator characteristic (ROC)-like analyses. These were then used for the entire cohort. MAIN RESULTS AND THE ROLE OF CHANCE Seventy-eight samples (71.5%) had results available from both STC and LTC; 12 samples (11%) had a result from STC only, and 7 samples (6.4%) had a result from LTC only. A chromosomal anomaly was detected in 55/86 (64%). The rates of chromosomal anomalies were 61, 72, 73 and 44% in patients undergoing their first, second, third and ≥4th pregnancy losses, respectively. The median cfDNA fetal fraction was 5%. With standard LLR thresholds used for noninvasive prenatal screening, the sensitivity of cfDNA in detecting aneuploidy was 55% (30/55) and with a specificity of 100% (31/31). Using pregnancy loss-specific LLR thresholds, the sensitivity of cfDNA in detecting aneuploidy was 82% (45/55), with a specificity of 90% (28/31). The positive and negative likelihood ratios were 8.46 and 0.20, respectively. Fetal sex was correctly assigned in all cases. LIMITATIONS, REASONS FOR CAUTION Cases with a false-positive result by cfDNA analysis would not receive the indicated RPL workup. Specificity could be improved by using a fetal fraction (FF) cutoff of 4%, but this would result in exclusion of more than a quarter of cases. WIDER IMPLICATIONS OF THE FINDINGS cfDNA-based testing can serve as an alternative to POC cytogenetic analysis and can guide further RPL management: if cfDNA demonstrates aneuploidy, no further action is taken and if no abnormality is detected, the recommended RPL workup is performed. STUDY FUNDING/COMPETING INTEREST(S) Cell-free DNA testing was funded by Illumina, Inc., San Diego, CA. Y.Y. is a member of Illumina’s Clinical Expert Panel and has received travel grants. A.B. has received travel grants from Illumina. All authors have no competing interest to declare.

The Balanced Scorecard (BSC) is a tool for strategic management that is used in many companies and organizations worldwide, both in the public and private sector. With this purpose it has also been used in healthcare organizations and institutions but there are not many studies on the implementation of BSC methodology in the day-to-day clinical laboratory. This review shows the strategy for the development of a BSC, which includes theoretical perspective objectives, as well as some indicators and goals with which the monitoring and quantitative measurement of the achievements of a strategic plan in a clinical laboratory can be done. Moreover, the results of the indicators allow the prioritization of the initiatives to be implemented each year. The methodology for the development of the proposed BSC includes the following steps: definition of theoretical objectives of each of the perspectives most used in the management of a clinical laboratory (customers, financial, internal processes and learning) taking into account the vision and the organizational model of the laboratory; creation of a strategic map of perspective objectives; definition of the relevant indicators to follow up on the objectives in a quantitative manner and establishment of the goals. Whether or not the laboratory is a reference laboratory, in which specific and infrequent analysis and health population programs are performed, is another fact to take into account. In this review a BSC for a reference clinical laboratory of the Spanish public sector is shown.

Recurrent pregnancy loss (RPL) is defined by ≥2 failed clinical pregnancies. The recommended workup (parental karyotyping, uterine cavity assessment, endocrine imbalances and autoimmune factors) will detect a probable cause in only ∼50% of cases. However, the most common cause of miscarriage, even in RPLs, is random chromosomal aberrations (trisomy, monosomy, triploidy). Chromosomal analysis of products of conception (POCs) has been shown to be cost-saving when used to guide further workup. This study was undertaken to assess whether maternal serum cell free (cf)DNA can be used to detect chromosomal abnormalities in RPLs. The study included 102 patients experiencing pregnancy loss at < 14 weeks. Blood samples were drawn for genome-wide cfDNA testing. Patients then underwent chorionic villous sampling (CVS) for karyotyping of both short-term and long-term cultures. Quantitative fluorescent PCR was also performed for some chromosomes. The first 50 cases served as a training set to determine appropriate thresholds for making an aneuploidy call. These were used for the entire cohort. The mean gestational age at embryo demise was 9.8 ± 2.1 weeks and the fetal fraction was 5.2 ± 3.7%. A chromosomal anomaly was detected in 68 cases. The rates of chromosomal anomalies were 67%, 74%, 73% and 42% in patients undergoing their 1st, 2nd, 3rd, and ≥4th losses, respectively. Of the 54 cases with a single autosomal trisomy or monosomy X, the detection rate was 83%. Of the 14 cases with complex abnormalities, monosomy, triploidy or mosaic, only 2 were detected. There were 4 false positive cases. The overall sensitivity of cfDNA-based testing was 69%. We suggest that cfDNA-based testing serve to guide management in RPLs: if cfDNA in the 2nd and subsequent RPL demonstrates aneuploidy, no further action is taken; if cfDNA demonstrates an unbalanced rearrangement, parental karyotypes is recommended; and if no abnormality is detected, then the recommended RPL workup is performed.

To explore a new molecular work-up based on the stepwise use of Quantitative Fluorescence Polymerase Chain Reaction (QF-PCR) extended to 9 chromosomes and SNP-array in chorionic villi retrieved by chorionic villi sampling (CVS) after routine offering to women with an early pregnancy loss. During a 3-year period (October 2015-October 2018), a new molecular work-up was prospectively assayed in chorionic villi, based on two QF-PCR rounds including the 21,18,13,7,X,Y,15,16 and 22 chromosomes, as the first step, and a high-resolution SNP-array as a second step. CVS was offered to all women with an early pregnancy loss before evacuation of the products of conception (POC), irrespectively of the number of previous losses, and routine genetic testing includes also karyotyping after short and long-term cultures. Among 459 women with an early pregnancy loss that consent to undergo CVS and the new work-up, chorionic villi were successfully retrieved in 454 (99%). In the first step, aneuploidies and triploidies were found in 227 (50%) cases including 3 complete uniparental disomies (complete moles). In the half of the losses with a normal QF-PCR result, routine karyotyping revealed 75 (17% of the total) atypical chromosomal anomalies, 15 (3.3%of the total) had an insufficient amount of remaining DNA, and 134 underwent SNP-arrays, revealing 5 (3.7%) pathogenic submicroscopic anomalies and 2 (1.4%) variants of unknown of significance. The use of a two-step molecular work-up in early pregnancy loss allowed to detect typical chromosomal anomalies in 50% of the cases, and 1.5% submicroscopic anomalies, in addition to 17% atypical chromosomal anomalies. Detection of maternal cell contamination, complete moles, and 3.7% failure rate are additional benefits over conventional karyotyping.

Objective: To assess whether the cisterna magna (CM) width measured in first-trimester fetuses is a useful marker for aneuploidy detection. Methods: This was a prospective study in 2 different cohorts in a tertiary referral center. The first cohort comprised 913 fetuses from the general pregnancy population during the period 2012–2016 and was used to construct the CM reference ranges applying the λ-μ-σ (LMS) method. The second cohort included 714 high-risk fetuses undergoing chorionic villus sampling during the period 2012–2016. Mean detection rates using the 95th percentile for CM width observed in chromosomal anomaly groups were compared with those obtained in chromosomally normal fetuses. Results: The 50th percentile for CM ranged from 1.66 to 2.75 mm when crown-rump length (CRL) increased from 45 to 84 mm. Among high-risk fetuses, the following chromosomal anomalies were diagnosed in 125 (17%) fetuses: trisomy 21 (n = 63), trisomy 18 or 13 (n = 21), monosomy X (n = 9), submicroscopic anomalies (n = 11), and other anomalies (n = 22). The mean CM width for euploid fetuses was 2.4 mm (1.13 multiples of the median, MoM). While CM width was significantly increased in trisomy 21 (mean 2.7 mm; 1.23 MoM; p > 0.05), no differences were found in the other anomaly groups. Among the 63 fetuses with trisomy 21, a CM width above the 99th percentile was observed in 23 fetuses (37%). Conclusions: The new reference range for CM width at 11–13 weeks of gestation did not differ from previous studies. In first-trimester fetuses with trisomy 21, CM width appears to be increased, although its value as an ultrasound marker is limited, because of its detection rate of 37%.

INTRODUCTION:For the measurands of the newborn screening (NBS), there are no analytical performance specifications (APS) available based on the Milan consensus Models. The objective is to provide total error (TE) APS based on the state-of-the-art (SOTA) for the NBS.MATERIAL AND METHODS:23,662 results were collected from the Spanish NBS EQA scheme between May 2015 and September 2018. Measurands included: thyroid-stimulating hormone (TSH), immunoreactive trypsinogen (IRT), phenylalanine (Phe), tyrosine (Tyr), free carnitine (C0), acetylcarnitine (C2), propionylcarnitine (C3), butyrylcarnitine (C4), isovalerylcarnitine (C5), glutarylcarnitine (C5DC), hexanoylcarnitine (C6), octanoylcarnitine (C8), decanoylcarnitine (C10), myristoylcarnitine (C14), palmitoylcarnitine (C16), stearoylcarnitine (C18). TE APS were calculated as the 90th percentile of the measurement errors, considering 75% of the best results from each laboratory only. It was also studied whether the analytical performance was concentration-dependent.RESULTS:When TE APS were calculated including all methods, TSH, IRT, C16 and C18 showed the best analytical performance and Phe, C5DC and C10 showed the worst. Generally, TE APS decreased when considering only majority methods and higher TE APS were obtained for lower concentrations.DISCUSSION:Due to the lack of APS based on superior models, the proposed TE APS based on the SOTA can help NBS laboratories to set quality specifications.

We report a case of spontaneous abortion associated with Zika virus infection in a pregnant woman who traveled from Spain to the Dominican Republic and developed a rash. Maternal Zika viremia persisted at least 31 days after onset of symptoms and 21 days after uterine evacuation.

In order to contribute to the knowledge of type and frequency of chromosome abnormalities in early pregnancy losses, we analyzed the cytogenetic results from a large series of first trimester miscarriages, using a diagnostic approach with a high success rate and no maternal contamination. A total of 1,119 consecutive chorionic villi samples were obtained before evacuation, and karyotypes were prepared after short-term culture (STC). In 603 samples, a long-term culture (LTC) was also performed. The overall and individual frequencies of the different types of chromosome abnormalities were established, including placental mosaicisms, and their relationship with maternal age and gestational weeks was assessed. An abnormal karyotype was detected in 70.3% of the samples. Single autosomal trisomy was the most frequent abnormality (64.6% of the abnormal cases), followed by triploidy (13.1%) and monosomy X (10.4%). Chromosome rearrangements were found in 5.2%, combined abnormalities in 8.9%, and placental mosaicism in 3.5% of the cases with STC and LTC performed. Individual trisomies behaved differently with respect to maternal age and intrauterine survival. Due to the combination of STC and LTC, our study offers reliable information on the incidence and type of chromosome abnormalities and placental mosaicism in miscarriages and contributes to define the cytogenetic implication in their etiology.

A new maternal age-dependent method to estimate absolute excess risks of trisomy 21, either after a previous trisomy 21 (homotrisomy) or after another trisomy (heterotrisomy), is proposed to be added to the estimated risk by conventional screening methods. Excess risk at term for a subsequent trisomy 21 was calculated from midtrimester risks reported by Morris et al., decreasing from 0.49% at 20 years to 0.01% at 46 years at the index pregnancy. Excess risk after a previous uncommon trisomy was derived from data reported by Warburton et al., decreasing from 0.37% at 20 years to 0.01% at 50 years.