bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Quantification of Human ABCA1 mRNA in Various Cell Types and Tissues by Real-Time Reverse Transcription-PCR
Authors: Kielar Danuta , Dietmaier Wolfgang , Langmann Thomas , Aslanidis Charalampos , Probst Mario , Naruszewicz Marek , Schmitz Gerd
Published: January 2020

Background: The ABCA1 gene encodes for a member of subfamily A of the ATP-binding cassette transporters that plays an important role in cellular export of cholesterol and phospholipids; therefore, quantification of the ABCA1 mRNA is critical in many studies related to its expression and regulation by metabolic factors, nutritional status, and new antiatherogenic drug candidates. We developed a rapid, sensitive, specific, and reproducible real-time reverse transcription-PCR (RT-PCR) method for detection and quantification of ABCA1 transcripts in total RNA isolated from cultured human cells and tissues.

Methods: To quantify ABCA1 mRNA, we generated a calibration curve from serial dilutions of in vitro-transcribed RNA corresponding to an amplified ABCA1 cDNA 205-bp fragment (homologous calibrator). Two pairs of fluorescent hybridization probes were used to detect the ABCA1 and porphobilinogen deaminase (PBGD) mRNAs; the latter served as an internal control. PCR was performed as real-time amplification of ABCA1 mRNA in 100 ng of total RNA isolated from various human tissues, and cultured cells were calculated from the calibration curve. In addition, normalized values of target (ABCA1/PBGD ratio) were calculated.

Results: Using this method, we quantified ABCA1 transcripts in various human tissue samples as well as in monocytes, THP-1 cells, fibroblasts, and adipocytes. We demonstrated ABCA1 mRNA up-regulation during human adipocyte and monocyte differentiation. In addition, we examined the effect of cholesterol loading and deloading on ABCA1 expression in monocytes, THP-1 cells, and fibroblasts.

Conclusions: Our RT-PCR assay allows the specific and highly reproducible detection and quantification of minute amounts of human ABCA1 mRNA. This new method is more accurate, more informative, and less laborious than the classic RT-PCR methods and Northern blot; it therefore could simplify all studies on ABCA1 mRNA expression.
Double-Gradient Denaturing Gradient Gel Electrophoresis Assay for Identification of L-Ferritin Iron-responsive Element Mutations Responsible for Hereditary Hyperferritinemia-Cataract Syndrome: Identification of the New Mutation C14G
Authors: Cremonesi Laura , Fumagalli Antonella , Soriani Nadia , Ferrari Maurizio , Levi Sonia , Belloli Silvana , Ruggeri Giuseppina , Arosio Paolo
Published: January 2020

Background: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis.

Methods: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5′ untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5–15% polyacrylamide and 50–95% denaturant) to separate the homo- and heteroduplexes.

Results: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin >400 μg/L. The new mutation G14C was identified.

Conclusions: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.
High-Throughput Genotyping of Thiopurine S-Methyltransferase by Denaturing HPLC
Authors: Schaeffeler Elke , Lang Thomas , Zanger Ulrich M , Eichelbaum Michel , Schwab Matthias
Published: January 2020

Background: The thiopurine S-methyltransferase (TPMT) genetic polymorphism has a significant clinical impact on the toxicity of thiopurine drugs, which are used in the treatment of leukemia and as immunosuppressants. To date, 10 mutant alleles are known that are associated with intermediate or low TPMT activity. To facilitate rapid screening of clinically relevant TPMT mutations, we developed a strategy of high-throughput genotyping by applying denaturing HPLC (DHPLC).

Methods: To test the specificity and efficiency of the DHPLC method, 98 DNA samples from a selected population of patients receiving thiopurine therapy or with previous thiopurine withdrawal were analyzed for the most frequent mutant TPMT alleles, *2 and *3A, which contain key mutations in exons 5, 7, and 10 to identify clearly different elution profiles. All fragments were examined by direct sequencing. Additionally, to test the sensitivity of DHPLC analysis, genotyping for the *2 and *3A alleles of all 98 DNA samples was performed by PCR-based methods (PCR-restriction fragment polymorphism analysis and allele-specific PCR).

Results: The presence of mutations discriminating for alleles *2, *3A, *3C, and *3D, as well as various silent and intron mutations, were correctly predicted by DHPLC in 100% of the samples as confirmed by direct sequencing. Comparison with PCR-based methods for alleles *2 and *3 produced an agreement of 100% with no false-negative signals.

Conclusions: DHPLC offers a highly sensitive, rapid, and efficient method for genotyping of the relevant TPMT mutations, discriminating at least for alleles *2 and *3, in clinical and laboratory practice. Additionally, DHPLC allows a simultaneous screening for novel genetic variability in the TPMT gene.
Preoperative Serum Prostate-specific Antigen (PSA) Below 10 μg/L Predicts Neither the Presence of Prostate Cancer Nor the Rate of Postoperative PSA Failure
Authors: Stamey Thomas A
Published: January 2020

Recent information on the relationship of serum prostate-specific antigen (PSA) to prostate cancer and new reports on death rates in men warrant a reassessment of how we diagnose and treat prostate cancer. We now know for the first time that the annual death rate from prostate cancer in men ≥65 years of age is only 226 per 100 000 men. At least 40 000 of 100 000 men over age 65 (40%) have invasive prostate cancer as judged by examination of prostates in 3- to 4-mm step-sections. Thus, only 1 of every 177 men 65 years of age or older (226 in 40 000) with invasive prostate cancer dies annually from his cancer. Serum PSA between 2 and 10 μg/L is used almost universally as an indication to biopsy the prostate. When 10–20 biopsies are commonly taken, it is not surprising that ∼40% of men are biopsy-positive for prostate cancer. Despite this reliance on serum PSA as an indication for biopsy, data at Stanford show no clinically useful relationship between preoperative serum PSA (in the range 2–10 mg/L) and the volume of Gleason grade 4/5 cancer or the volume of Gleason grades 3, 2, and 1 cancer, nor can we show any useful relationship of such preoperative PSA concentrations (2–10 μg/L) to biochemical PSA failure rates after radical prostatectomy. We urgently need a better serum marker for prostate cancer. Because PSA biochemical failure rates after radical prostatectomy are directly proportional to the amount of Gleason grade 4/5 cancer in the prostate, a serum marker of Gleason grade 4/5 carcinoma could be ideal.
Improved Diagnostic Classification of Alcohol Abusers by Combining Carbohydrate-deficient Transferrin and γ-Glutamyltransferase
Authors: Sillanaukee Pekka , Olsson Ulf
Published: January 2020

Background: Biochemical markers can provide objective evidence of high alcohol consumption. However, currently available markers have limitations in their diagnostic performance.

Methods: The diagnostic values of the most frequently used markers [carbohydrate-deficient transferrin (CDT), γ-glutamyltransferase (GGT), aspartate aminotransferase, alanine aminotransferase, and mean corpuscular volume] were studied in an analysis of six different clinical studies (n = 1412) on alcohol abusers and social drinkers. The purpose of the analyses was to determine whether a combination of markers would improve the diagnosis of subjects.

Results: Discrimination between alcohol abusers and social drinkers, as measured by the areas under nonparametric ROC plots, was significantly better (P <0.001) for the new combined marker [γ-CDT = 0.8 · ln(GGT) + 1.3 · ln(CDT)] than for any of the separate markers or combination of CDT or GGT with other markers. The cutoff values for γ-CDT (6.5) can be taken to be the same among males and females.

Conclusions: The combined variable γ-CDT is a powerful tool to discriminate alcohol abusers from social drinkers and is recommended for clinical use.
Fatty Acid Ethyl Esters in Liver and Adipose Tissues as Postmortem Markers for Ethanol Intake
Authors: Salem Raneem O , Refaai Majed A , Cluette-Brown Joanne E , Russo Joshua W , Laposata Michael
Published: January 2020

Background: Fatty acid ethyl esters (FAEEs) are nonoxidative metabolites of ethanol. FAEEs are found in liver, pancreas, and adipose tissues up to 24 h after consumption of ethanol, and on that basis, they are potentially useful markers for ethanol intake. In this study with rats, we investigated the efficacy of using FAEEs in liver and in adipose tissue as postmortem markers for premortem ethanol ingestion.

Methods: An animal study was conducted in which test rats received injections of ethanol and control rats received injections of normal saline. The rats were killed 2 h after the injections. The bodies of the animals were stored at 4 °C up to 12 h, and samples of liver and adipose tissues were collected at different time intervals and processed for FAEE quantification. In another set of experiments, the rats received injections and were killed as described above, but bodies of animals from both groups were stored at 4, 25, or 37 °C for up to 72 h, and liver samples were collected and processed for FAEE quantification.

Results: FAEEs were detected up to 12 h after death in liver and adipose tissue samples from the bodies of ethanol-treated animals stored at 4 °C; negligible amounts were detected in the bodies of animals that received normal saline. Adipose tissues contained higher amounts of FAEEs than liver, as well as more species: eight FAEE species in adipose tissue and five in liver tissue. Higher concentrations of FAEEs were detected in livers of treated animals stored at 25 °C for up to 48 h than in livers of controls stored under the same conditions.

Conclusions: For at least 12 h after death, FAEEs in liver and adipose tissues are useful postmortem markers of premortem ethanol ingestion.
Semiautomated DNA Mutation Analysis Using a Robotic Workstation and Molecular Beacons
Authors: Smit Maarten L , Giesendorf Belinda A J , Vet Jacqueline A M , Trijbels Frans J M , Blom Henk J
Published: January 2020

Background: Our increasing knowledge of the genetic basis of inheritable diseases requires the development of automated reliable methods for high-throughput analyses.

Methods: We investigated the combination of semiautomated DNA extraction from blood using a robotic workstation, followed by automated mutation detection using highly specific fluorescent DNA probes, so-called molecular beacons, which can discriminate between alleles with as little as one single-base mutation. We designed two molecular beacons, one recognizing the wild-type allele and the other the mutant allele, to determine genotypes in a single reaction. To evaluate this procedure, we examined the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, which is associated with an increased risk for cardiovascular disease and neural tube defects. DNA was isolated from 10 μL of fresh EDTA-blood samples by use of a robotic workstation. The DNA samples were analyzed using molecular beacons as well as conventional methods.

Results: Both methods were compared, and no differences were found between outcomes of genotyping.

Conclusions: The described assay enables robust and automated extraction of DNA and analysis of up to 96 samples (10 μL of blood per sample) within 5 h. This is superior to conventional methods and makes it suitable for high-throughput analyses.
Pharmacokinetic Basis for the Efficient and Safe Use of Low-Dose Mycophenolate Mofetil in Combination with Tacrolimus in Kidney Transplantation
Authors: Mourad Michel , Malaise Jacques , Chaib Eddour Djamila , De Meyer Martine , König Josiane , Schepers Raf , Squifflet Jean Paul , Wallemacq Pierre
Published: January 2020

Background: Mycophenolate mofetil (MMF) is an effective posttransplantation immunosuppressive agent used in combination with cyclosporin A (CsA) or tacrolimus (Tc). An increase in plasma mycophenolic acid (MPA) has been shown in patients receiving Tc-MMF combination therapy compared with CsA-MMF combination therapy at the same dose of MMF. The aim of this prospective study was to assess the pharmacokinetic/pharmacodynamic (PK/PD) relationship for MPA in kidney transplant patients receiving low-dose MMF (500 mg twice a day) in combination with Tc.

Methods: Adult kidney transplant recipients (n = 51) were included. MPA-PK profiles (blood sampling at 0, 0.5, 1, 2, 4, 6, and 12 h after MMF oral dose) were obtained within the first 2 weeks after transplantation, 3 months after grafting, and at every adverse clinical event [side effect or acute rejection (AR)]. All patients received Tc, MMF (500 mg twice a day), and steroids.

Results: Thirty patients (59%) had uneventful outcomes, and 21 patients had 33 episodes of MPA-related side effects; only 3 patients had AR. A total of 78 MPA-PK profiles were obtained. The following PK parameters were increased in the side-effects group compared with the non-side effects group: mean MPA cmin, 2.63 ± 1.58 vs 1.75 ± 0.82 mg/L (P = 0.016); mean c30, 10.47 ± 6.27 vs 7.66 ± 8.95 mg/L (P = 0.009); mean c60, 9.67 ± 5.42 vs 5.83 ± 2.6 mg/L (P = 0.0002); mean area under the MPA time-concentration curve from 0 to 12 h [MPA-AUC(0–12)], 48.38 ± 18.5 vs 36.04 ± 10.82 mg · h/L (P = 0.0006); mean dose-normalized MPA-AUC, 0.16 ± 0.05 vs 0.12 ± 0.04 (mg · h/L)/(mg/m2) (P = 0.0015). For the three AR patients, MPA concentrations obtained at the time of AR revealed MPA cmin values of 1.86, 1.76, and 3.83 mg/L, respectively, and MPA-AUC(0–12) values of 37.7, 24.9, and 104.9 mg · h/L. The threshold of toxicity was 3 mg/L (sensitivity, 38.7%; specificity, 91.5%) for cmin, 8.09 mg/L for maximum MPA concentration during the first hour (sensitivity, 77.8%; specificity, 67.4%), and 37.6 mg · h/L for MPA-AUC(0–12) (sensitivity, 83.3%; specificity, 59.6%).

Conclusions: These results demonstrate the relationship between plasma MPA concentrations and toxicity. High cmin, c30, and c60 values as well as AUC(0–12) are associated with increased risk for side effects. These values may have an importance in a routine monitoring program.
Vitamin B12 Treatment Normalizes Metabolic Markers But Has Limited Clinical Effect: A Randomized Placebo-controlled Study
Authors: Hvas Anne-Mette , Ellegaard Jørgen , Nexø Ebba
Published: January 2020

Background: The clinical significance of increased plasma methylmalonic acid (P-MMA) is unclear. We assessed the efficacy of vitamin B12 treatment in reducing P-MMA and plasma total homocysteine compared with the clinical benefits of treatment.

Methods: We studied 140 individuals with mildly to modestly increased P-MMA (0.40–2.00 μmol/L), not previously treated with vitamin B12, in a randomized, placebo-controlled study. A detailed medical history was obtained, and laboratory tests as well as an objective neurologic disability score were performed at baseline and 3 months after the start of intervention.

Results: P-MMA (P <0.001) or plasma total homocysteine (P <0.001) decreased in the treatment group vs the placebo group, but no significant difference was found in the change of blood hemoglobin (P = 0.18) and mean cell volume (P = 0.71). Changes in symptom scores did not differ between the groups for symptoms of anemia (P = 0.63), neurologic symptoms (P = 0.21), gastroenterologic symptoms (P = 0.32), or the Neurological Disability Score (P = 0.85).

Conclusions: Treatment with vitamin B12 reduces P-MMA and plasma total homocysteine, but individuals with a mild to modest increase in P-MMA may have only limited clinical benefit from vitamin B12 treatment, at least in the short term.
Clinical Evaluation of the Elecsys β-CrossLaps Serum Assay, a New Assay for Degradation Products of Type I Collagen C-Telopeptides
Authors: Okabe Reiko , Nakatsuka Kiyoshi , Inaba Masaaki , Miki Takami , Naka Hiroshi , Masaki Hideki , Moriguchi Atushi , Nishizawa Yoshiki
Published: January 2020

Background: The Elecsys β-CrossLaps serum assay measures type I collagen degradation fragments (β-CTx) that contain the β-isomerized octapeptide EKAHD-β-GGR. We investigated the analytical performance of the assay and changes in β-CrossLaps in patients with metabolic bone diseases.

Methods: The electrochemiluminescent sandwich immunoassay uses two monoclonal antibodies directed against different regions of the linear EKAHD-β-GGR.

Results: β-CrossLaps (β-CTx) immunoreactivity was stable in serum and plasma stored at 4 °C for 24 h or at room temperature for 4 h, and it did not decrease appreciably in samples stored at −30 °C for 12 weeks. Nine cycles of repeated freezing-thawing did not affect serum β-CTx. The intra- and interassay imprecision (CVs) for four samples was ≤2.6% (n = 10) and ≤4.1% (n = 10), respectively. The mean day-to-day biological variation (CV) was 20% in 10 postmenopausal women (n = 10 days). Serum β-CTx and osteocalcin were correlated in patients with hyperparathyroidism (r = 0.796; P <0.0001; n = 28), chronic renal failure on hemodialysis (r = 0.784; P = 0.0003; n = 16), hypoparathyroidism (r = 0.950; P = 0.0001; n = 11), and pseudohypoparathyroidism (r = 0.987; P = 0.130; n = 4). Serum β-CTx decreased by 47.4% ± 8.8% (mean ± SD) and 60.7% ± 6.5% at 3 and 6 months, respectively, after initiation of estrogen replacement therapy in 34 women. These decreases were greater than the decreases in urinary excretion of deoxypyridinoline (31.8% ± 3.9% and 38.1% ± 4.4%, respectively) or pyridinoline cross-linked C-terminal telopeptide of type I collagen (15.9% ± 3.9% and 16.9% ± 4.6%, respectively).

Conclusions: The Elecsys β-CrossLaps serum assay provides a potentially useful tool for assessing bone resorption state, including its response to estrogen replacement therapy.
Overview of Evidence-based Medicine: Challenges for Evidence-based Laboratory Medicine
Authors: McQueen Matthew J
Published: January 2020

Evidence-based medicine (EBM) has been driven by the need to cope with information overload, by cost-control, and by a public impatient for the best in diagnostics and treatment. Clinical guidelines, care maps, and outcome measures are quality improvement tools for the appropriateness, efficiency, and effectiveness of health services. Although they are imperfect, their value increases with the quality of the evidence they incorporate. Laboratory professionals must direct more effort to demonstrating the impact of laboratory tests on a greater variety of clinical outcomes. Laboratory and clinical practitioners must be familiar with many of the accessible electronic and paper tools for searching for evidence. Detailed statistical and epidemiologic knowledge is not essential, but critical appraisal skills and a competent understanding of the strengths and weaknesses of systematic review and metaanalysis are necessary. Overemphasis on complexity and failure to recognize time limitations are major barriers to translating EBM into everyday practice. Emphasizing and practicing the role of the laboratory professional as a skilled clinical consultant strongly grounded in evidence as well, in addition to better integration of laboratory and clinical information and improved laboratory reports will overcome most barriers. There is a poverty of good, primary studies of test evaluations. Institution of more consistent standards for the design and reporting of studies on diagnostic accuracy should improve the situation. If nothing else, systematic reviews have demonstrated the need for more good-quality primary research in laboratory medicine.
Diagnosis of Trisomy 21 in Fetal Nucleated Erythrocytes from Maternal Blood by Use of Short Tandem Repeat Sequences
Authors: Samura Osamu , Sohda Satoshi , Johnson Kirby L , Pertl Barbara , Ralston Steven , Delli-Bovi Laurent C , Bianchi Diana W
Published: January 2020

Background: The purpose of this study was to determine whether aneuploid fetal nucleated erythrocytes (NRBCs) could be detected in maternal blood through the use of fluorescent PCR amplification with polymorphic short tandem repeat (STR) markers as an alternative or complementary method to analysis by fluorescent in situ hybridization (FISH).

Methods: Peripheral blood samples were obtained from women who had just undergone termination of pregnancy because of fetal trisomy 21 (three cases, 47,XY,+21; four cases, 47,XX,+21). Candidate fetal cells were isolated by flow-sorting by antibodies to the γ chain of fetal hemoglobin and Hoechst 33342. FISH analysis was performed by the use of chromosome-specific probes for X, Y, and 21. Fetal NRBCs, as defined by the presence of γ staining, characteristic morphology, and three chromosome 21 signals, along with maternal leukocytes, defined as γ negative and two chromosome 21 signals, were micromanipulated separately and subjected to fluorescent PCR amplification of chromosome 21 STR markers (D21S11, D21S1411, and/or D21S1412).

Results: In five of seven cases analyzed, fetal NRBCs were aneuploid, as determined by the presence of triallelic or diallelic peaks of chromosome 21 sequences when compared with sequences from the maternal leukocytes.

Conclusions: Fluorescent PCR amplification of STRs can detect fetal aneuploidy and may be useful in the setting of poor hybridization efficiency with FISH analysis. These results suggest that combined fetal aneuploidy and single-gene diagnoses by the use of DNA microarrays may be feasible in the near future.
Complete Scanning of the Hereditary Hemochromatosis Gene (HFE) by Use of Denaturing HPLC
Authors: Le Gac Gerald , Mura Catherine , Férec Claude
Published: January 2020

Background: Between 4% and 35% of hereditary hemochromatosis (HC) probands are C282Y or H63D heterozygotes or lack both of these two common HFE mutations, and 15 novel HFE mutations have been described recently. We evaluated denaturing HPLC (DHPLC) for screening of the whole HFE coding region and further defined whether HC probands with an incomplete HFE genotype carry uncommon mutations.

Methods: Analytical conditions for each coding exon were determined by a combination of computer melting profile predictions and experimental melting curves. To test accuracy for scanning the complete HFE coding region and optimize DHPLC running conditions, each melting domain was investigated with at least one mutation or one polymorphism as reference. We tested 100 DNA samples harboring the C282Y, H63D, or S65C mutations and 17 artificially created positive controls that carried either 1 of the 14 other known HFE mutations or 3 selected polymorphisms.

Results: Investigations on each of the coding exons 1, 2, 4, 5, and 6 could be performed at one analysis temperature. Coding exon 3 displayed a more complex melting profile and required two analysis temperatures. DHPLC detected all known HFE mutations as well as the three selected polymorphisms.

Conclusions: DHPLC can be used to scan the HFE gene in HC probands in whom at least one chromosome lacks an assigned mutation.
Comparative Costs of Treating Adults and Children within Selected Diagnosis-related Groups
Authors: Young Donald S , Sachais Bruce S , Jefferies Leigh C
Published: January 2020

Background: There have been no large-scale analyses of resource utilization comparing the overall costs to treat pediatric patients vs adult patients. Likewise, there have been no studies evaluating the costs of the various components of hospitalization (e.g., accommodation, laboratory, radiology, and drugs) among adult and pediatric populations.

Methods: To study the effect of age on the costs of treating patients, we have evaluated 43 conditions with matching diagnosis-related groups (DRGs) for children and adults. Using a database developed by the University HealthSystems Consortium, we examined the major non-physician components of hospital costs, including accommodation, surgery, pharmacy, radiology, and laboratory for 1 346 028 patient admissions to 60 University hospitals. These costs were derived from the ratio of costs to charges based on the Centers for Medicare and Medicaid Services PPS UB-2 cost reports.

Results: The total non-physician cost of treating adults was generally greater than that for children within paired DRGs. Some of this difference may be attributable to the overall longer stay of adults in hospital. For conditions that were nominally the same, radiology, laboratory, and drug costs, especially tended to be higher for adults than for children. This was most marked when the costs were evaluated on a per diem basis. There tended to be greater variability in the costs of treating children than adults within the paired DRGs, as evidenced by greater differences between the median and mean costs.

Conclusions: Among University hospitals, the costs of managing children are typically less than for adults with the same nominal condition. In these hospitals, there tends to be less use of laboratory, radiology, and pharmacy services for children than for adults.
High-Throughput Detection of Submicroscopic Deletions and Methylation Status at 15q11-q13 by a Photo-Cross-Linking Oligonucleotide Hybridization Assay
Authors: Peoples Risa , Weltman Helena , Van Atta Reuel , Wang Jingli , Wood Michael , Ferrante-Raimondi Marta , Cheng Peter , Huan Bingfang
Published: January 2020

Background: Current technologies for assessing genetic deletions and duplications of greater than one kilobase are labor-intensive or rely on PCR-based methods, and none offers the ability to simultaneously detect dosage abnormalities, assess 5′-to-3′ cytosine-guanosine (CpG) methylation, and interrogate single-nucleotide polymorphisms (SNPs). We describe a high-throughput platform for direct gene-dosage determination capable of concurrent assessment of other forms of gene modification.

Methods: We used a light-activated interstrand nucleic acid cross-linking system (XLntTM technology) to determine gene dosage at the 15q11-q13 deletion/duplication locus. We incorporated restriction enzyme digestion of genomic DNA into the method to assess CpG methylation in parallel with gene dosage. For method validation we used DNA from 31 cell lines with previously characterized 15q11-q13 gene dosage and parental origin status. Diagnostic cutoffs were set at 0.5 ± 0.15, 1 ± 0.15–0.25, and 2 ± 0.3.

Results: Dosage-only experiments showed discrimination of deletions (n = 21) from healthy controls (NCs; n = 27) in all samples. Five of 49 samples gave results outside of specification. Concurrent evaluation of dosage and CpG methylation yielded dosage results within specification for 18 of 19 deletion and 8 of 12 NC samples. Paternal deletion and NC methylation pattern results were within specification in 17 of 19 and 9 of 12 runs, respectively. No overlap was demonstrated between value sets for the two groups.

Conclusions: The XLnt technology provides a rapid, high-throughput platform for the accurate determination of gene dosage. The flexibility of this technology allows parallel interrogation of gene dosage, CpG methylation, and SNPs.
European Concerted Action on Anticoagulation. Use of Plasma Samples to Derive International Sensitivity Index for Whole-Blood Prothrombin Time Monitors
Authors: Poller Leon , Keown Michelle , Chauhan Nikhil , van den Besselaar Anton MHP , Meeuwisse-Braun Joyce , Tripodi Armando , Clerici Mariagrazia , Jespersen Jørgen
Published: January 2020

Background: To simplify International Sensitivity Index (ISI) calibration, the possibility of substituting fresh plasma for fresh whole-blood samples with point-of-care testing (POCT) whole-blood monitors was investigated in a three-center study of three different POCT systems.

Methods: A modified full WHO calibration procedure based on 20 healthy controls and 60 coumarin-treated patients was performed on three monitoring systems with whole-blood and plasma samples against plasma tested using the European Concerted Action on Anticoagulation (ECAA) rabbit reference plain thromboplastin and the manual prothrombin time (PT) method.

Results: With one of the three systems, the mean ISI was 1.51 for whole blood and 1.49 for plasma; with the second system, the mean ISI was 1.08 for both whole blood and plasma. With the third system, however, the difference between the mean ISI for whole blood and that for plasma was greater (1.15 and 1.01, respectively). Overall, the precision of the calibrations was less than with traditional manual plasma PT testing.

Conclusions: Provided that an appropriate calcium chloride concentration is used, the plasma PT results can be used for accurate ISI calibration of two of these three whole-blood POCT systems. Precision criteria need to be modified for POCT monitors.
Preservation of Urine for Flow Cytometric and Visual Microscopic Testing
Authors: Kouri Timo , Vuotari Lotta , Pohjavaara Simo , Laippala Pekka
Published: January 2020

Background: Preservatives that could prevent destruction of cells, casts, and bacteria in urine are of great practical importance because they allow centralization and improvement of accuracy of urine particle counting. We compared two in-house mixtures and one commercial solution, as well as refrigeration, for their ability to preserve urine for both automated analysis (flow cytometry) and visual microscopy.

Methods: Urine specimens were preserved by refrigeration at 4 °C without preservatives (procedure 1); in a lyophilized solution intended to preserve specimens for bacterial culture (Urine C&S tubes; BD Preanalytical Solutions; procedure 2); in 10 mL/L formalin–0.15 mol/L NaCl (procedure 3); in 80 mL/L ethanol–20 g/L polyethylene glycol (procedure 4); and by storage at 20 °C without preservatives (procedure 5). Test strip measurements were used to select specimens positive for leukocyte esterase, hemoglobin, albumin, or nitrite. For 106 consecutive strip-positive specimens, urinalysis was performed by UF-100TM (Sysmex) and by phase-contrast microscopy after Sternheimer supravital staining. Automated analysis was performed at arrival in the morning, on the same day in the afternoon, and after 1 and 3 days. Visual microscopy was performed at arrival and 3 days later.

Results: Urine bacterial counts were well preserved with procedures 1–3, with a false-positive rate of 0.0–3.4% at day 3 vs 28% without preservation (procedure 5). Erythrocytes were poorly preserved for 3 days (κ coefficients, 0.24–0.61); after 1 day, fair preservation was seen with procedure 2 (κ = 0.78), compared with less favorable preservation with procedure 1 (κ = 0.61) or procedure 5 (κ = 0.66). Leukocytes were well preserved by all five procedures in the acidic adult urines investigated. Counts of casts and large epithelial cells were artifactually increased by procedure 3. Procedure 2 performed at least as well as refrigeration for specimens analyzed with visual microscopy.

Conclusions: Urine specimens from adults can be stabilized at room temperature for both automated particle analysis and visual microscopy.
Application of Microarrays to the Analysis of Gene Expression in Cancer
Authors: Macgregor Pascale F , Squire Jeremy A
Published: January 2020

Molecular diagnostics is a rapidly advancing field in which insights into disease mechanisms are being elucidated by use of new gene-based biomarkers. Until recently, diagnostic and prognostic assessment of diseased tissues and tumors relied heavily on indirect indicators that permitted only general classifications into broad histologic or morphologic subtypes and did not take into account the alterations in individual gene expression. Global expression analysis using microarrays now allows for simultaneous interrogation of the expression of thousands of genes in a high-throughput fashion and offers unprecedented opportunities to obtain molecular signatures of the state of activity of diseased cells and patient samples. Microarray analysis may provide invaluable information on disease pathology, progression, resistance to treatment, and response to cellular microenvironments and ultimately may lead to improved early diagnosis and innovative therapeutic approaches for cancer.
The Spectrophotometric Determination of the Oxygen Saturation of Blood
Authors: Johnston George W , Holtkamp Freddy , Eve John R
Published: January 2020

A modification of the Gordy and Drabkin method for the determination of oxygen saturation of blood has been presented. The equipment used is readily available, and the results are sufficiently accurate for clinical use. The method is particularly useful in analysing multiple samples of blood such as obtained by cardiac catheterization. Approximately 500 blood samples have been analysed by the described procedure during a 6-month period. Ten samples may easily be analysed during a 1-hour period using the described procedure.
Measurement of Isocitric Dehydrogenase Activity in Body Fluids
Authors: Bowers George N
Published: January 2020

1. Factors influencing the isocitric dehydrogenase (ICD) activity of serum were studied. Substrate inhibition by high concentrations of isocitrate was observed.

2. The temperature-activity curve of serum ICD was determined. The importance of controlling the temperature of the reaction was stressed.

3. Using this information, a method of measuring the ICD activity in 0.2 ml. of serum is described.

4. The range of ICD activity in 50 healthy males was 30 to 192 units with a mean of 90 units and a standard deviation of ± 34.3 units.
A Scheme for the Rapid Identification in Urine of Commonly Used Sedatives, Hypnotics, and Tranquilizers
Authors: Sobolewski Georges , Nadeau Guy
Published: January 2020

A procedure is described to identify common sedatives, hypnotics, and ataractics in 20 ml. of urine, with the aid of four extractions and five relatively simple reactions. The whole scheme, which involves no special equipment and requires about 2 hours, will detect the drugs at the usual therapeutic levels and, hence, has been found convenient not only in acute drug intoxication, but also in ascertaining that patients comply with the prescribed medication.
Study on the Determination of Heavy Water in Plasma or Urine
Authors: Gaebler O H , Choitz Harold C
Published: January 2020

A technic has been described for determining the excess of deuterium oxide in plasma or urine, by directly measuring 19:18 ratios in vapor from these fluids with a mass spectrometer of suitable design. Although the 19:18 ratio for water varied with sample size and other experimental conditions, the increment in ratio per unit concentration of added D2O remained quite uniform. A virtually linear relationship between the increment in ratio and the concentration of added D2O also facilitated checking the calibration. The time required for flushing out deuterium when a Pyrex inlet system was used could be minimized by carrying out both the calibrations and the analyses in triplicate, without intermediate flushing, but with adherence to a strict time schedule. Results calculated from increments in ratio for the second and third samples of vapor rarely differed from their average value by more than ± 0.8 per cent.
Simplified Technic for Estimation of Serum Lipoprotein Cholesterol
Authors: Searcy R L , Bergquist L M , Jung R C , Craig R , Korotzer J
Published: January 2020

A phosphomolybdic acid reagent was used to identify serum alpha- and beta-lipoproteins separated electrophoretically on filter paper. Cholesterol associated with these areas on unstained strips was eluted directly into a ferrous sulfate-acetic acid mixture for colorimetric analysis. The results obtained with the proposed method compared favorably with those of Nury and Smith (7). The sums of the values obtained for the alpha- and beta-lipoprotein cholesterols agreed well with measurements of serum total cholesterol by another method (12). Sex and age differences in the concentration of alpha- and beta-lipoprotein cholesterol were suggested by results obtained on 200 randomly selected blood donors.
Simplified Determination of the Lipid Components of Blood Serum
Authors: Connerty Harold V , Briggs Anglis R , Eaton Edward H
Published: January 2020

A simplified scheme of analysis is presented for the determination of free and esterified cholesterol, the phospholipids, and the neutral fats (triglycerides) of blood serum. The hydroxamic acid reaction is used to determine total esterified fatty acids. The phospholipids are determined after acid digestion of a trichloroacetic acid precipitate of protein-phospholipid, and both total and free cholesterol are determined by a modified ferric ammonium sulfate reagent. Complete details of methods and calculations are described and discussed.
Unusual Electrophoretic Patterns of Plasma Proteins in Human Subjects
Authors: Kanabrocki Eugene L
Published: January 2020

Study of 313 plasma electrophoretic patterns from 158 hospitalized subjects revealed that 30 per cent of these patients exhibited a heterogeneity marked by occurrence of double peaks in the alpha-2 and 6 per cent in the beta globulin fraction. Occurrence of double peaks could not be related to any specific disease. Similar study of plasma from 18 normal adult subjects did not reveal double peaks in any of the protein fractions. Existence of prealbumin components, with mobilities greater than that of the albumin, has been observed in 6 of 158 pathological subjects. Mobilities and concentrations of components x1, x2, and x3 are reported.
X-Ray Spectroscopy in the Clinical Laboratory
Authors: Natelson Samuel , Sheid Bertrum
Published: January 2020

X-ray spectroscopy was applied to the determination of the total phosphorus content of serum and the iron content of whole blood (hemoglobin). Samples were placed on confined spots on paper, dried, then passed through the X-ray field. Concentration was then determined from the peaks as drawn on a recorder. A device is described that permits automatic assay of successive samples dried on paper without delays between readings. Samples are placed in the field and rapidly withdrawn at 30-sec. intervals, the response of the recorder serving as a measure of concentration. Results for total phosphorus in serum and total iron in whole blood were compared with those obtained by a wet ashing procedure. No significant difference with respect to both mean values and precision was observed except that total iron by the wet ashing procedure was more precise. However, the X-ray technic was adequate for clinical purposes. Hemoglobin levels calculated from whole-blood iron values demonstrated that bilirubin, red cell debris, leukocytes, and lipids interfered in the colorimetric but not in the X-ray procedure. A hemoglobinometer based on the X-ray spectroscopic technic may, therefore, yield results of greater clinical significance.
Simplified Technic for the Determination of Serum Protein-Bound Iodine
Authors: Faulkner Louise W , Levy Richard P , Leonards Jack R
Published: January 2020

A simplified technic for the determination of serum protein-bound iodine (PBI) is presented. Endogenously labeled l131 PBI is completely recovered. The technic is based on the method of Barker et al. (1) and includes the following modifications: The number of washes of the precipitated serum proteins are decreased. Unashed iodide standards are used in a sodium carbonate solution rather than an internal standard. A temperature-correction formula is employed to allow the cericarsenite-iodide reaction to be carried out at room temperature, and a stable cerate color is produced with brucine sulfate so that the colorimetry is carried out with greater convenience. The serum PBI concentrations are read directly from a graph, which simplifies the calculations.
Absorption Measurements for Oxygenated and Reduced Hemoglobin in the Range 0.6—1.88 Microns
Authors: Barlow Robert B , Polanyi Michael L
Published: January 2020

Absorbance measurements are made for oxygenated and reduced hemoglobin in the range 0.6—1.88 µ for application to new methods of dye-dilution studies. Experimental results show that an isosbestic region exists between 1.3 and 1.88 µ, with the relative extinction coefficients of O2Hb and Hb equaling zero at 1.3, 1.67, and 1.77 µ.
Continuous In-Vivo Estimation of Blood Alcohol
Authors: Nadeau Guy , Fortin Benoit , Dugal Pierre
Published: January 2020

Automated chemical analysis was applied to constant in-vivo experiments on dogs for the determination of blood alcohol. The continuous blood collection was similar to that in earlier experiments for determination of blood glucose in humans.

Although interferences by glucose was observed in the chemical methodology used, preliminary data show that the rise of blood alcohol is slower following I.V. administration of alcohol-water mixtures than with alcohol-saline solutions. With the experimental animal under pentobarbital anesthesia, the alcohol equilibrium is reached more rapidly and the elimination period is significantly prolonged.
Use of The Sanz Pipet in the Clinical Laboratory
Authors: Westover Leola , Wertman Maxine , Ware Arnold G
Published: January 2020

The Sanz micropipet, which is made of polyethylene tubing, is evaluated. With use of the chloride determination (Cotlove Chloridometer) as a reference analysis, it is shown that the pipetting error is well under 1 per cent.
Dehydrogenase of Regenerating Red Blood Cells in Phenylhydrazine Anemia
Authors: Friedman Max M , Kahn Bernard S , Lapan Bernard
Published: January 2020

Dehydrogenase activities in red blood cells of rabbits made anemic with phenylhydrazine were assayed. These assays were continued at the time of reticulocytosis and followed through the course of in vivo maturation of the reticulocytes to a younger erythrocyte population.

Isocitric, glucose-6-phosphate, and 6-phosphogluconic dehydrogenases were more active in the reticulocytes. Isocitric and glucose-6-phosphate dehydrogenases were also more active in the younger red cell, compared to an average population. Lactic dehydrogenase activity was the same in the reticulocyte, young red cell, and the older red cell.

The significance of these findings is discussed.
A Punched-Card System for the Laboratory
Authors: Radin Nathan
Published: January 2020

A punched-card system was developed to simplify the administrative aspects of the clinical chemistry laboratory. In it a three-part requisition and samples are matched and given a laboratory number. The third copy of the original laboratory requsition is a charge card also used as a name file for the day. Prepunched cards, coded for laboratory tests, are used for each test requested. The laboratory number is stamped and punched on each test card. All test cards are then sorted into laboratory test groups, after which the laboratory data and results are recorded on them. Cards are then sorted for laboratory number and rematched with the original laboratory requisitions, which remained in numerical order. Results are then copied directly onto the requisitions.

The system is more efficient, less susceptible to error, and simpler than other laboratory record systems.
A New Diazo Method for the Determination of Bilirubin
Authors: Rand Royden N , Pasqua Alfred di
Published: January 2020

A new technic for the determination of total bilirubin is reported. The substitution of 2,4 dichloroaniline for sulfanilic acid results in a very stable diazotized reagent which reacts quickly with bilirubin in plasma or serum. Comparison with a sulfanilic acid procedure indicates that similar analytic values are obtained with this technic; short quality control studies indicate satisfactory precision was obtained.
Colorimetric Method for Hippuric Acid
Authors: Umberger Charles J , Fiorese Frank F
Published: January 2020

Hippuric acid dissolved in pyridine producesa red-orange color upon the addition of benzenesulfonyl chloride. The color has a stable absorbance at 380 mµ. It is sensitive in microgram quantities of hippuric acid and follows the Beer-Lambert Law over a wide range of concentrations. The mechanism of the color reaction is described.
Spinal-Fluid Protein Determination for the Differentiation of Neurologic Disorders
Authors: Papadopoulos N M , Hess W C , O'Doherly D , Wakeman L
Published: January 2020

A simple chemical test previously described for the determination of total protein and -globulin was applied to the analysis of spinal fluid specimens (CSF) from non-neurologic and a variety of neurologic disorders. On the basis of the chemical findings, the pathologic specimens analyzed were divided into three groups: (a) nonneurologic and some neurologic conditions with a normal CSF total protein and -globulin concentration; (b) demyelinating disorders with a normal total protein and elevated b.gamma;-globulin concentration; and (c) degenerative and infectious with an elevated total protein and -globulin. A good correlation between chemical findings and clinical observation was obtained.
Macro and Micro Methods for the Determination of Serum Iron and Iron-Binding Capacity
Authors: Caraway Wendell T
Published: January 2020

Blood serum is incubated with ascorbic acid and hydrochloric acid to liberate protein-bound iron. Proteins are precipitated with trichloroacetic acid in the presence of chloroform. To an aliquot of clear supernatant is added a solution of tripyridyltriazine and sufficient ammonium acetate to adjust the ρH between 4 and 5. Absorbance of the blue-colored complex is measured at 590 m µ . Iron-binding capacity is measured by saturation of serum with ferric iron followed by removal of excess iron with magnesium carbonate. Measurement of total bound iron is completed as above. Macro and micro technics for either determination require 2 ml. and 0.1 ml. of serum, respectively. Reliability of the methods has been established by recovery experiments, replicate analyses, and variation of reagent concentrations and incubation times.
Automation of Protein-Bound Iodine Determinations
Authors: Stevens Clarence O , Levandoski Nicholas G
Published: January 2020

Portions of the Barker alkali-incineration technic for determining protein-bound iodine have been automated using an automatic analysis system. The manifold and analytic procedure are described in detail. Data are presented to show that the automated procedure compares favorably in accuracy with the manual procedure.
Relationship of Serum and Urinary Proteins in Neoplastic States: A Preliminary Survey
Authors: Nettleship A , Strother James A , Smith H Siemsen
Published: January 2020

Twenty-four-hour urine specimens from patients with and without malignancies were concentrated by dialysis. The electrophoretic mobility of the urine protein components was compared with that of the respective serum proteins. Control values were similar to those established in the literature. Urinary protein concentrations found in malignant states were higher. Generally, there is a positive correlation between serum concentration of globulins and urinary excretion. It is not possible to prove in either controls or in patients bearing malignancies that the amount of urinary protein excreted depends upon molecular size.