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We established several 5'-nuclease PCR assays (sequence-specific priming and fluorogenic probing) for detection of fetal Rhesus D-specific DNA. When using EDTA-anticoagulated blood samples from pregnant women we were able to analyse fetal DNA in maternal plasma for prediction of fetal Rhesus D blood group. Methods: Primer and probe sets were established using nucleotide differences between the RHD and RHCE gene within exon 3 & 7, the 3'-terminal part of exon 10, and the insertion/deletion region in intron 4. With these four sets a maximum of hybrid D/CE genes can be determined by PCR (e.g. DIIIb, DIVa, DVII, DVI, DDBT, DDFR, -D-, CD-, etc.). Four separate primer mixes and dye-conjugated hybridization probes (reporter dye=5'-FAM; quencher dye=3'-TAMRA) were optimised for specificity and sensitive detection of RHD-amplicons. Results: (1) We investigated the accuracy of both PCR assays with 54 different pretyped DNA samples from D-negative, D-positive individuals and our rare donor panel. All samples were 100% concordant typing results (blinded and unblinded study). (2) Three rare partial D-species [DIVa, DVI, DDFR] were used for dilution experiments in RhD(-) DNA (range 0,01-10%). RhD(+) DNA could be detected at the lowest level of 0,2%. (3) Samples drawn at gestation week 28-41 from 14 pregnancies have been analyzed and compared with newborn samples. 8 of 8 D-negative samples and 6 of 6 D-positive samples were detected and concordant with serologic results. Conclusion: We established a quantitative detection assay for RhD+ DNA naturally occurring in plasma of pregnant women. This assay has the potential for sensitive RhD diagnosis in peripheral blood samples and can be used for non-invasive detection of a D-positive fetus in a D-negative pregnant woman, thereby avoiding diagnostic puncture (amniocentesis) of the fetus and the risk of antibody boostering in the last trimester. [ABSTRACT FROM AUTHOR]

Objectives: Marked physiological changes in growth and development present challenges in defining pediatric reference intervals for biomarkers of health and disease. Lambda, Mu, and Sigma (LMS)-based statistical modeling provides a continuous normal distribution by negating skewness and variation, and is commonly used to establish growth charts. Such LMS reference curves are suggested to enhance laboratory test result interpretation. The current study establishes LMS-based continuous reference percentiles for 14 biomarkers in the CALIPER cohort of healthy children and adolescents., Methods: Data from healthy children and adolescents aged 1-<19 years were used to establish continuous reference percentiles using a novel LMS-based statistical method, including 2.5th, 25th, 50th, 75th, and 97.5th percentiles. The LMS approach applies a Box-Cox data transformation and summarizes continuous distributions by age via three curves: skewness (Lambda), median (Mu), and coefficient of variation (Sigma)., Results: LMS-based percentiles and z-scores were generated for 14 common pediatric biomarkers that demonstrate dynamic concentration patterns with age (e.g., alkaline phosphatase) and/or wherein the magnitude of difference from the population mean may be clinically relevant (e.g., triglycerides). The LMS model captured age- and sex-specific distributions accurately and was not substantially influenced by outlying points., Conclusions: This is the first study to establish LMS-based continuous reference percentiles for biochemical markers in a healthy Canadian pediatric population. The current LMS-based approach builds upon previous continuous reference interval models by providing graded percentiles to improve test result interpretation, particularly with repeated measures over time. This method may assist in facilitating a patient-centered approach to laboratory medicine., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

Context: Hormone reference intervals in pediatric endocrinology are traditionally partitioned by age and lack the framework for benchmarking individual blood test results as normalized z-scores and plotting sequential measurements onto a chart. Reference curve modeling is applicable to endocrine variables and represents a standardized method to account for variation with gender and age., Objective: We aimed to establish gender-specific biomarker reference curves for clinical use and benchmark associations between hormones, pubertal phenotype, and body mass index (BMI)., Methods: Using cross-sectional population sample data from 2139 healthy Norwegian children and adolescents, we analyzed the pubertal status, ultrasound measures of glandular breast tissue (girls) and testicular volume (boys), BMI, and laboratory measurements of 17 clinical biomarkers modeled using the established "LMS" growth chart algorithm in R., Results: Reference curves for puberty hormones and pertinent biomarkers were modeled to adjust for age and gender. Z-score equivalents of biomarker levels and anthropometric measurements were compiled in a comprehensive beta coefficient matrix for each gender. Excerpted from this analysis and independently of age, BMI was positively associated with female glandular breast volume (β = 0.5, P < 0.001) and leptin (β = 0.6, P < 0.001), and inversely correlated with serum levels of sex hormone-binding globulin (SHBG) (β = -0.4, P < 0.001). Biomarker z-score profiles differed significantly between cohort subgroups stratified by puberty phenotype and BMI weight class., Conclusion: Biomarker reference curves and corresponding z-scores provide an intuitive framework for clinical implementation in pediatric endocrinology and facilitate the application of machine learning classification and covariate precision medicine for pediatric patients., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.)

Recent studies suggest that both high and low levels of vitamin B12 (vitB12) may have negative health impacts. We measured VitB12 in patients with the Neurodevelopmental disorders (ND) (n = 222), comprised of Autism Spectrum Disorders, specific Developmental disorders, and Intellectual Disability (aged 2-53 years), schizophrenia (n = 401), and healthy controls (HC) (n = 483). Age-and gender-adjusted vitB12 z-scores were calculated by comparisons with a reference population (n = 76 148). We found higher vitB12 in ND (median 420 pmol/L, mean z-score: 0.30) than in HC (316 pmol/L, z-score: 0.06, P < .01) and schizophrenia (306 pmol/L, z-score: -0.02, P < .001), which was significant after adjusting for age, gender, vitB12 supplement, folate, hemoglobin, leukocytes, liver, and kidney function (P < .02). In ND, 20% (n = 44) had vitB12 above 650 pmol/L, and 1% (n = 3) had below 150 pmol/L (common reference limits). In 6.3% (n = 14) of ND, vitB12 was above 2SD of mean in the age-and gender-adjusted reference population, which was more frequent than in HC (n = 8, 1.6%), OR: 4.0, P = .001. Low vitB12 was equally frequent as in HC, and vitB12 z-scores were equal across the age groups. To conclude, vitB12 was higher in ND than in HC and schizophrenia, suggesting a specific feature of ND, which warrants further studies to investigate the underlying mechanisms., (© 2020 Federation of American Societies for Experimental Biology.)

Background: The microbial detection system BacT/ALERT (bioMérieux) is widely used to monitor bacterial contamination of platelet concentrates (PCs). Recently, the manufacturer introduced polycarbonate culture bottles and a modified pH-sensitive liquid emulsion sensor as microbial growth indicator. This reconfigured assay was investigated in a routine setting., Study Design and Methods: In each of eight transfusion centers, samples from 500 consecutive PCs were monitored for 1 week. For all PCs with a positive BacT/ALERT signal, retained samples and, if available, original PC containers and concomitant red blood cell concentrates were analyzed independently. Initially BacT/ALERT-positive PCs without bacterial identification in any sample were defined as false-positive. BacT/ALERT-positive PCs with bacteria in the first sample only were called potentially positive. PCs with bacteria in the first sample and the same strain in at least one additional sample were accepted as positive., Results: Five PCs (0.13%) were positive, 9 PCs (0.23%) were potentially positive, and 35 PCs (0.9%) were false-positive. The rate of false-positive BacT/ALERT results varied substantially between centers (<0.2%-3.2%). Tracings from false-positive cultures lacked an exponential increase of the signal during incubation. Most of these false-positives were due to malfunctioning cells in various BacT/ALERT incubation units., Conclusion: Careful assessment of individual tracings of samples with positive signals helps to identify malfunctioning incubation units. Their early shutdown or replacement minimizes the high rate of unrectifiable product rejects attributed to false-positive alarms and avoids unnecessary concern of doctors and patients after conversion to a reconfigured BacT/ALERT assay.

In contrast to the success achieved with the production of hybridomas in the mouse system, creating human hybridomas is problematic. The reason is believed to be a lack of suitable malignant human cell lines. The work presented here demonstrates the establishment of three human parent cell lines--two of which are of T cell origin--by installing hypoxanthine guanosine phosphoribosyl transferase (HGPRT) and/or thimidine kinase (TK) deficiencies into the leukemic cell lines REH, 1301 and SKW-3. In order to isolate true hybridomas, selection procedures must guarantee complete death of the enzyme-deficient malignant parent cells. In this respect sublines with a combined HGPRT and TK deficiency proved to be superior to those with only one enzyme deficiency, especially in combination with the newly developed hypoxanthine/aminopterine/thymidine/azaserine (HATA) selection medium. However, selection media should be of low nonspecific toxicity. This was shown to be a particular property of the thymidine-free azaserine/hypoxanthine (AH) selection medium. Preliminary data show an extraordinary ability of one subclone of the hypoxanthine guanosine phosphoribosyl transferase-negative T cell line SKW-3 to generate human T-T hybridomas. They are of a stable, nearly tetraploid karyotype and express new surface antigens, thus providing new possibilities for the investigation of human T lymphocyte function by means of hybridoma technology.

With the intention of reducing the psychological stress and anxiety of patients during their admission to the medical intensive care unit, specially selected and prepared instrumental music was played over a loud speaker to the patient cubicles. The patients' opinions on the programme and their subjective state of health were determined by a questionnaire. 78% of the patients felt that their well-being was improved by the music. With the exception of 7%, who were disturbed by the music, all other patients found it reassuring, diverting, hypnotic or entertaining. In addition to the patients 11 doctors and 16 nurses from the intensive care unit as well as 38 doctors and 80 nurses from 39 medical intensive care units in German university clinics and large hospitals were asked for their opinions on the use of music for intensive care patients.