Comparison of three different protocols for obtaining hemolysis.

OBJECTIVES: Hemolysis is associated with erroneous or delayed results. Objectives of the study were to compare four different methods for obtaining hemolysis in vitro on three different analyzers. METHODS: Hemolysis was prepared with addition of pure hemoglobin into serum pool, osmotic shock, aspiration through blood collection needle, freezing/thawing of whole blood. Biochemistry parameters were measured in duplicate at Architect c8000 (Abbott, Abbott Park, USA), Beckman Coulter AU680 (Beckman Coulter, Brea, USA) and Cobas 6000 c501 (Roche, Mannheim, Germany), according to manufacturers' declarations. Cut-off value was defined as the highest value of H index with corresponding bias lower than acceptance criteria. RESULTS: We were not able to obtain results with freezing protocol. On all three platforms, lowest number of analytes were sensitive to hemolysis at H=0.5 using method of adding free hemoglobin. When osmotic shock was used, cut-off values for the most analytes were generally met at lower values. Hemolysis significantly interfered with measurement of potassium and lactate dehydrogenase (LD) at H=0.5 on all platforms. The most of the tested analytes had the lowest acceptable H index when aspiration method was used. At the low level of hemolysis (H=0.8) glucose, sodium, potassium, chloride, phosphate, and LD were affected on all analyzers, with some additional analytes depending on the manufacturer. CONCLUSIONS: Hemolysis interference differs on different analyzers and according to protocol for obtaining hemolysis. Aspiration method was generally the most sensitive to hemolysis interference, while addition of free Hb was the most resistant.

Association of polymorphisms in promoter region of TNF-α -238 and -308 with clinical outcomes in patients with immune-mediated inflammatory diseases on anti-TNF therapy.

The hypothesis of the study was that polymorphisms in promoter regions -238 and -308 of TNF-α could be associated with different clinical outcomes in inflammatory bowel diseases (IBD) and immune-mediated rheumatic diseases (IMRD). The aim was to examine the possible association of both polymorphisms with concentration of C-reactive protein (CRP) and fecal calprotectin (fCAL), onset of the remission and development of the ADA in patients on therapy with anti-TNF inhibitors. The prospective study was done in patients with IBD and IMRD on infliximab (IFX) or adalimumab (ADM). Patients were genotyped for TNF-α -238 and -308 polymorphisms. The concentration of CRP, fCAL, IFX or ADM and antibodies to drugs were measured according to manufacturer's instructions and followed-up for 6 or 12 months. Out of all patients (N = 112), number of patients in remission did not differ according to genotypes (for IBD patients P = 0.509 vs 0.223; for IMRD patients P = 0.541 vs 0.132 for TNF-α -238 and -308, respectively). Initial CRP concentration was higher in IBD patients with TNF-α -308 GG than GA/AA genotypes in patients who failed to achieve remission [11.8 (4.4-39.6) vs 3.1 (1.5-6.5), P = 0.033]. In IBD patients with remission, fCAL concentration after at least 6 months of therapy was higher in TNF-α-308 GG than in GA genotype [52 (25-552) vs 20 (20-20) µg/g, P = 0.041]. Our results showed the association of TNF-α -308 GG genotype with a higher concentration of CRP and fecal calprotectin in patients with inflammatory bowel diseases on IFX or ADM therapy. Clinical remission and development of antibodies to anti-TNF drugs were not associated with TNF-α -238 and -308 polymorphisms.

Lower concentration of vitamin D is associated with lower DAS28 and VAS-pain scores in patients with inflammatory rheumatic diseases treated with infliximab: a pilot study.

Vitamin D is beneficial in patients with immune-mediated rheumatic diseases as it has been shown that it lowers the incidence risk and the level of inflammation. To examine the association between clinical outcomes and initial 25-hydroxyvitamin D [25(OH)D] concentrations in patients with the immune-mediated rheumatic diseases treated with infliximab for 9 months. This study was performed in patients with rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) treated with infliximab for at least 38 weeks. Disease activity was assessed using Disease Activity Score (DAS28) for RA and PsA and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) for AS, while the global assessment was performed using the Visual Analogue Scale (VAS). Patients were divided into 2 groups according to 25(OH)D concentration which was classified as deficient or non-deficient (below and above 50 nmol/L, respectively). Concentrations of infliximab (IFX) and C-reactive protein (CRP) were measured according to the manufacturer's instructions.This study was performed in patients with rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) treated with infliximab for at least 38 weeks. Disease activity was assessed using Disease Activity Score (DAS28) for RA and PsA and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) for AS, while the global assessment was performed using the Visual Analogue Scale (VAS). Patients were divided into 2 groups according to 25(OH)D concentration which was classified as deficient or non-deficient (below and above 50 nmol/L, respectively). Concentrations of infliximab (IFX) and C-reactive protein (CRP) were measured according to the manufacturer's instructions. The study included 23 patients (14 with RA, 6 with AS and 3 with PsA), median age 54 years, 15 females. Vitamin D deficient and non-deficient groups had median initial concentrations of 38 and 61 nmol/L, respectively. DAS28 and pain on VAS calculated at the 2nd and 38th week showed a statistically significant decrease only in RA and PsA patients with vitamin D deficiency (P = 0.02 and 0.06, respectively). Lower initial concentration of 25(OH)D in patients treated with infliximab was associated with better improvement of clinical measures (DAS28 and VAS) of disease after 9 months of therapy.

Integrity of serum samples is changed by modified centrifugation conditions.

Background Serum samples should be centrifuged for at least 10 min at 1300-2500 × g. Changed centrifugation conditions could compromise sample quality. The objective of this study was to compare the serum quality and turnaround time (TAT) using different centrifugation conditions. Methods The study was done in four different periods (A, B, C and D) at different conditions: for 10, 5 and 7 (A, B and C, respectively) at 2876 × g, and 7 (D) min at 4141 × g. Sample quality was assessed as the proportion of samples with: (a) aspiration errors, (b) H index >0.5 g/L and (c) suppressed reports of potassium (K) due to hemolysis. TAT was calculated for emergency samples. The proportions of samples (a), (b) and (c) were compared according to period A. Results The number of aspiration errors was significantly higher in samples centrifuged at 2876 × g for 5 min (p = 0.021) and remained unchanged when centrifuged for 7 min (p = 0.066 and 0.177, for periods C and D, respectively). In periods B, C and D, the proportion of samples with hemolysis was higher than that in period A (p-values 0.039, 0.009 and 0.042, respectively). TAT differed between all periods (p < 0.001), with the lowest TAT observed for B and D. The lowest number of samples exceeding 60-min TAT was observed in period D (p = 0.011). Conclusions The integrity of serum samples is changed with different centrifugation conditions than those recommended. Our study showed that shorter centrifugation at higher force (7 min at 4141 × g) significantly decreases TAT, with unchanged proportion of samples with aspiration errors.

Genetic and lifestyle predictors of ischemic stroke severity and outcome.

BACKGROUND: Different models that include clinical variables and blood markers have been investigated to predict acute ischemic stroke treatment course and recovery. AIM: The aim of the study was to investigate associations between lipid levels, lifestyle factors, hemostatic (F5, F2, SERPINE1, F13A1, and FGB), and atherogenic (APOA5 and ACE) gene variants and acute ischemic stroke (AIS) severity. MATERIALS AND METHODS: This study included 250 patients with AIS in which F5, F2, SERPINE1, F13A1, FGB, APOA5, and ACE genotypes were determined. Total cholesterol (TC), high-density cholesterol, low-density cholesterol, and triglycerides concentrations were measured within 24 h of the AIS onset. Examination of the neurological deficit was done using National Institutes of Health Stroke Scale/Score (NIHSS). RESULTS: APOA5 genotype [TC + CC] was more frequent (P = 0.026) in patients with the NIHSS score ≥ 21. Univariate regression analysis has shown that triglycerides (OR 0.55, 95% CI 0.34-0.91; P = 0.019), obesity (0.28, 95% CI 0.10-0.73; P = 0.010), age (OR 1.08, 95% CI 1.04-1.13; P < 0.001), and APOA5 genotype (TC + CC) (OR 2.40, 95% CI 1.10-5.25; P = 0.034) are significantly associated with a severe stroke. When all variables were included in model age (OR 1.06, 95% CI 1.01-1.11; P = 0.018), obesity (OR 0.25, 95% CI 0.08-0.77; P = 0.016) and APOA5 genotype (TC + CC) (OR 3.26, 95% CI 1.29-8.23; P = 0.012) remained significant for the risk of severe AIS. CONCLUSION: APOA5 genotype (TC + CC), age, and obesity could be used as prognostic risk factors for a very severe stroke (NIHSS ≥ 21).

Visual assessment of hemolysis affects patient safety.

BACKGROUND: Manual handling of hemolyzed samples is not standardized and is vulnerable to errors. This study aimed to evaluate laboratory errors due to manual handling of hemolyzed samples and to assess the risk they might have for patient safety. METHODS: Data were retrospectively obtained from a laboratory information system for 25 emergency tests from hemolyzed samples. Hemolysis (concentration of free hemoglobin >0.5 g/L) was visually assessed by comparison with a color chart. The reference person reestimated the routinely assessed degree of hemolysis to all samples (n=3185) received in the laboratory in a 1-week period. For each test, the correct and incorrect way of handling results was determined. Risk assessment was performed according to ISO 14971 standard with five categories of risk (S1-S5) and error occurrence (O1-O5). RESULTS: In the studied period, the emergency laboratory received 495 hemolyzed samples (15.5%) with a total of 2518 laboratory test requests (15.5%): 102 (20.6%) of the reports from hemolyzed samples had a comment on hemolysis; 31% of the test results were handled incorrectly (20.7% due to the incorrect release of the test result despite hemolysis interference and 10.3% due to unnecessary suppression), accounting for 4.8% of the total test volume. Tests with the highest combination of risk and occurrence rate were troponin T, potassium and total bilirubin. CONCLUSIONS: Manual handling of hemolyzed samples may lead to risk of errors in reporting results for troponin T, potassium and total bilirubin, which may have an effect on clinical decision. In addition, unnecessary suppression of the sample results unaffected by hemolysis could affect patient outcome.

Precision, accuracy, cross reactivity and comparability of serum indices measurement on Abbott Architect c8000, Beckman Coulter AU5800 and Roche Cobas 6000 c501 clinical chemistry analyzers.

BACKGROUND: The aim of our study was to perform verification of serum indices on three clinical chemistry platforms. METHODS: This study was done on three analyzers: Abbott Architect c8000, Beckman Coulter AU5800 (BC) and Roche Cobas 6000 c501. The following analytical specifications were verified: precision (two patient samples), accuracy (sample with the highest concentration of interferent was serially diluted and measured values compared to theoretical values), comparability (120 patients samples) and cross reactivity (samples with increasing concentrations of interferent were divided in two aliquots and remaining interferents were added in each aliquot. Measurements were done before and after adding interferents). RESULTS: Best results for precision were obtained for the H index (0.72%-2.08%). Accuracy for the H index was acceptable for Cobas and BC, while on Architect, deviations in the high concentration range were observed (y=0.02 [0.01-0.07]+1.07 [1.06-1.08]x). All three analyzers showed acceptable results in evaluating accuracy of L index and unacceptable results for I index. The H index was comparable between BC and both, Architect (Cohen's κ [95% CI]=0.795 [0.692-0.898]) and Roche (Cohen's κ [95% CI]=0.825 [0.729-0.922]), while Roche and Architect were not comparable. The I index was not comparable between all analyzer combinations, while the L index was only comparable between Abbott and BC. Cross reactivity analysis mostly showed that serum indices measurement is affected when a combination of interferences is present. CONCLUSIONS: There is heterogeneity between analyzers in the hemolysis, icteria, lipemia (HIL) quality performance. Verification of serum indices in routine work is necessary to establish analytical specifications.

Patient's knowledge and awareness about the effect of the over-the-counter (OTC) drugs and dietary supplements on laboratory test results: a survey in 18 European countries.

Background Nowadays over-the-counter (OTC) drugs and dietary supplements are widely used. Their use can have a significant impact on the validity of laboratory results. The aim of this multicenter European study was to determine the frequency of consumption of various dietary products and OTC drugs among patients and explore their level of knowledge and awareness about the potential impact of various products on laboratory test results. Methods Eighteen European countries participated in this study. The survey was carried out anonymously on a subsequent series of outpatients (n=200) in each participating country. Included were patients who were referred to the laboratory for blood sampling and who voluntarily agreed to participate in the study. The survey included questions about the frequency of consumption of various products, awareness of the importance of informing physicians and laboratory staff about it and information about influence of preanalytical factors in general on laboratory test results. Results In total, 68% of patients were regularly taking at least one OTC drug or dietary supplement. The frequency of patients consuming at least one OTC drug or dietary supplement differed between countries (p=0.001). Vitamins (38%), minerals (34%), cranberry juice (20%), acetylsalicylic acid (ASA) (17%) and omega fatty acids (17%) were the most commonly used in our study. Conclusions The use of various OTC drugs and dietary supplements is highly prevalent in Europe and patients are often not willing to disclose this information to the laboratory staff and ordering physician. The education of both patients and healthcare staff is needed.

Ascorbic acid-A black hole of urine chemistry screening.

BACKGROUND: Study was performed in order: (i) to assess the comparability of glucose, bilirubin, hemoglobin, leukocyte esterase, and protein; (ii) to assess accuracy of glucose, bilirubin, hemoglobin, leukocyte esterase, and protein; and (iii) to evaluate interference of ascorbic acid on the glucose, bilirubin, hemoglobin, and nitrite determination using 2 different dipsticks: iChem Velocity, Iris Diagnostics and Combur-10M, Roche Diagnostics. METHODS: Random urine specimens were included in the study. Comparability, accuracy, and ascorbic acid interference testing were performed. RESULTS: Obtained results have shown almost perfect agreement for all parameters between 2 dipsticks in samples with negative ascorbic acid. Agreement in samples with positive ascorbic acid was not acceptable for bilirubin, protein, nitrite, and hemoglobin. Accuracy was not acceptable for hemoglobin and leukocyte esterase on both dipsticks. Ascorbic acid interference examination has shown that intensity of interference differs between dipsticks. Ascorbic acid interferes with glucose, hemoglobin, nitrite, and bilirubin at different concentrations causing false-negative results. CONCLUSION: Obtained results indicate that it is necessary to determine diagnostic accuracy of used dipstick in order to define purpose of urinalysis. It is very important to choose dipstick with ascorbic acid indicator and to examine ascorbic acid impact on dipstick analytes independently of manufacturer claims.

Key Performance Indicators to Measure Improvement After Implementation of Total Laboratory Automation Abbott Accelerator a3600.

The aim of the study was to estimate improvement of work efficiency in the laboratory after implementation of total laboratory automation (TLA) by Abbott Accelerator a3600 in the laboratory with measuring different key performance indicators (KPIs) before and after TLA implementation. The objective was also to recommend steps for defining KPIs in other laboratories. For evaluation of improvement 10 organizational and/or technical KPIs were defined for all phases of laboratory work and measured before (November 2013) and after (from 2015 to 2017) TLA implementation. Out of 10 defined KPIs, 9 were successfully measured and significantly improved. Waiting time for registration of samples in the LIS was significantly reduced from 16 (9-28) to 9 (6-16) minutes after TLA (P < 0.001). After TLA all tests were performed at core biochemistry analyzers which significantly reduced walking distance for sample management (for more than 800 m per worker) and number of tube touches (for almost 50%). Analyzers downtime and engagement time for analyzers maintenance was reduced for 50 h and 28 h per month, respectively. TLA eliminated manual dilution of samples with extreme results with sigma values increment from 3.4 to >6 after TLA. Although median turnaround time TAT for potassium and troponin was higher (for approximately 20 min), number of outliers with TAT >60 min expressed as sigma values were satisfying (>3). Implementation of the TLA improved the most of the processes in our laboratory with 9 out of 10 properly defined and measured KPIs. With proper planning and defining of KPIs, every laboratory could measure changes in daily workflow.

Reactogenicity and Peak Anti-RBD-S1 IgG Concentrations in Individuals with No Prior COVID-19 Infection Vaccinated with Different SARS-CoV-2 Vaccines.

OBJECTIVE: To investigate the association of immune response with vaccination adverse effects at peak anti-receptor-binding domain spike subunit 1 (anti-RBDS1) IgG after full vaccination with Comirnaty, Spikevax, or Vaxzevria. METHODS: Anti-RBDS1 IgG concentrations after vaccination were determined in healthy adults vaccinated with the Comirnaty, Spikevax, and Vaxzevria vaccines. The association of reactogenicity and peak antibody response after vaccination was tested. RESULTS: Anti-RBDS1 IgG values were significantly higher in the Comirnaty and Spikevax group, compared with the Vaxzevria group (P < .001). Fever and muscle pain were found to be significant independent predictors of peak anti-RBDS1 IgG in the Comirnaty and Spikevax groups (P = .03 and P = .02, respectively). The multivariate model, adjusted for covariates, showed that no association between reactogenicity and peak antibody concentrations was found in the Comirnaty, Spikevax, and Vaxzevria groups. CONCLUSIONS: No association between reactogenicity and peak anti-RBDS1 IgG after vaccination with the Comirnaty, Spikevax, and Vaxzevria vaccine was found.

Serum Calprotectin Is a Valid Biomarker in Distinction of Bacterial Urinary Tract Infection From Viral Respiratory Illness in Children Under 3 Years of Age.

Background: Febrile illnesses in young children can be a major diagnostic challenge, despite the routine use of various laboratory markers. Recent advancements in the understanding of inflammatory processes have highlighted the role of calprotectin, a heterodimer consisting of S100A8 and S100A9 proteins, with many studies suggesting its clinical value as a biomarker of inflammation. This research aimed to evaluate the usefulness of serum calprotectin (sCal) as a biomarker of urinary tract infection (UTI), which was due to its high pooled prevalence and feasibility of urine culture as a diagnostic reference standard selected for a model of bacterial infection in children. Methods: Febrile children aged 0-36 months with suspected UTI based on positive urinalysis or viral respiratory tract infection were included. Children with significant bacteriuria in urine culture were labeled as cases (n = 58), while those with confirmed viral infection (n = 51), as well as those with suspected UTI but sterile urine culture who went on to develop symptoms consistent with viral respiratory infection (n = 7), were labeled as controls. sCal levels were determined by a commercial immunoassay. Conventional inflammation markers (C-reactive protein, procalcitonin, white blood cell count, absolute neutrophil count, and neutrophil percentage) were measured on the day of the clinical examination. Differences in measured inflammatory markers between cases and controls were analyzed with Mann-Whitney U-test. ROC analysis reported cut-off values with the best sensitivity and specificity to distinguish bacterial UTI from viral respiratory infection. Results: All analyzed inflammatory biomarkers, including sCal, were significantly higher in cases than in controls. Median concentration of sCal was 4.97 µg/mL (IQR 3.43-6.42) and 2.45 µg/mL (IQR 1.63-3.85) for cases and controls, respectively (p < 0.001). For identifying bacterial UTI, sensitivity and specificity of sCal were 77.6 and 69.0%, respectively, at an adjusted cut-off point of >3.24 µg/mL (AUC 80.2%). Conclusion: sCal could have substantial added value in the management of a child with fever and positive urinalysis and is a promising biomarker in distinction between bacterial UTI and viral respiratory causes of febrile illness in children under the age of 3 years.

Saliva Changes in Pediatric Patients with Eating Disorders.

Objectives: To determine the differences between pediatric patients with eating disorders (ED) and the control group in the amount of saliva and the concentration of total amylase and electrolytes in saliva, and to evaluate the correlation between the saliva changes and nutritional status. Material and methods: The study included 101 participants (14.34 ±1.99 years), out of which 50 participants with ED subgroups and 51 participants in the control group. Data were statistically analyzed (Mann-Whitney, Kruskal-Wallis, chi-square, Spearman rank correlation test, α=0.05). Results: No significant differences in salivary volume between the groups were found. A significant difference in the volume of saliva secreted in the 5th and 15th minute was found between the anorexia nervosa and bulimia nervosa subgroups. The examined anthropometric parameters were marginally or significantly positively associated with saliva volume at 5 and 15 minutes, noting a more significant correlation of the same at 15 than at 5 minutes. The patients with ED had a significantly higher concentration of inorganic phosphates in saliva while the concentrations of other electrolytes and total amylase in saliva did not differ significantly. Conclusions: Nutritional status affects salivation. There is a difference in saliva volume in pediatric patients with different ED disorders. Variations in saliva electrolytes in pediatric patients with ED are possible.

Haemoglobin A1c-based screening for prediabetes and diabetes mellitus: a multi-center study in Croatian adult population.

INTRODUCTION: Based on the hypothesis that there is a substantial rate of adults with prediabetes and undiagnosed diabetes mellitus (DM), our aim was to perform haemoglobin A1c (HbA1c)-based screening in a cohort of Croatian adults and estimate the prevalence of prediabetes and undiagnosed DM according to American Diabetes Association criteria. MATERIALS AND METHODS: This multi-center, cross-sectional study performed in six Croatian hospitals included 5527 patients aged 40 to 70 years admitted to the Emergency Department or undergoing a primary care check-up. Haemoglobin A1c was measured from leftover whole blood samples using the enzymatic method on either Alinity c or Architect c-series analyser (Abbott Laboratories, Chicago, USA). Haemoglobin A1c between 39-47 mmol/mol was classified as prediabetes, while ≥ 48 mmol/mol as undiagnosed DM. RESULTS: After exclusion of 435 patients with known DM, the final cohort included 5092 patients (median age 57; 56% males). A total of 882 (17.3%) patients had HbA1c values between 39 and 47 mmol/mol. There were 214 (4.2%) patients with HbA1c ≥ 48 mmol/mol. Prediabetes prevalence ranged from 14.2% to 20.5%, while undiagnosed DM from 3.3% to 7.3%, with statistically significant differences among settings (P < 0.001). Age-stratified analysis showed that prediabetes and undiagnosed DM prevalence increase with age (P < 0.001), being 25.4% and 5.8%, respectively, in patients aged 60 to 70 years. CONCLUSION: Underlying impairment of glucose metabolism was identified in about one in five adults, with significant number of patients with already overt DM. These results should serve as a starting point for further steps directed towards promotion of preventive measures for DM in Croatia.

Laboratory policies and practices for thyroid function tests in Croatia: survey on behalf of Working Group for Laboratory Endocrinology of the Croatian Society of Medical Biochemistry and Laboratory Medicine.

Introduction: Laboratory plays important part in screening, diagnosis, and management of thyroid disorders. The aim of this study was to estimate current laboratory preanalytical, analytical and postanalytical practices and policies in Croatia. Materials and methods: Working Group for Laboratory Endocrinology of the Croatian Society of Medical Biochemistry and Laboratory Medicine designed a questionnaire with 27 questions and statements regarding practices and protocols in measuring thyroid function tests. The survey was sent to 111 medical biochemistry laboratories participating in external quality assurance scheme for thyroid hormones organized by Croatian Centre for Quality Assessment in Laboratory Medicine. Data is presented as absolute numbers and proportions. Results: Fifty-three participants returned the questionnaire. Response rate varied depending on question, yielding a total survey response rate of 46-48%. All respondents perform thyroid stimulating hormone (TSH). From all other thyroid tests, most performed is free thyroxine (37/53) and least TSH-stimulating immunoglobulin (1/53). Laboratories are using nine different immunoassay methods. One tenth of laboratories is verifying manufacturer's declared limit of quantification for TSH and one third is verifying implemented reference intervals for all performed tests. Most of laboratories (91%) adopt the manufacturer's reference interval for adult population. Reference intervals for TSH are reported with different percentiles (90, 95 or 99 percentiles). Conclusion: This survey showed current practices and policies in Croatian laboratories regarding thyroid testing. The results identified some critical spots and will serve as a foundation in creating national guidelines in order to harmonize laboratory procedures in thyroid testing in Croatia.

Impact of Preanalytical and Analytical Methods on Cell-Free DNA Diagnostics.

While tissue biopsy has for the longest time been the gold-standard in biomedicine, precision/personalized medicine is making the shift toward liquid biopsies. Cell-free DNA (cfDNA) based genetic and epigenetic biomarkers reflect the molecular status of its tissue-of-origin allowing for early and non-invasive diagnostics of different pathologies. However, selection of preanalytical procedures (including cfDNA isolation) as well as analytical methods are known to impact the downstream results. Calls for greater standardization are made continuously, yet comprehensive assessments of the impact on diagnostic parameters are lacking. This study aims to evaluate the preanalytic and analytic factors that influence cfDNA diagnostic parameters in blood and semen. Text mining analysis has been performed to assess cfDNA research trends, and identify studies on isolation methods, preanalytical and analytical impact. Seminal and blood plasma were tested as liquid biopsy sources. Traditional methods of cfDNA isolation, commercial kits (CKs), and an in-house developed protocol were tested, as well as the impact of dithiothreitol (DTT) on cfDNA isolation performance. Fluorimetry, qPCR, digital droplet PCR (ddPCR), and bioanalyzer were compared as cfDNA quantification methods. Fragment analysis was performed by qPCR and bioanalyzer while the downstream application (cfDNA methylation) was analyzed by pyrosequencing. In contrast to blood, semen as a liquid biopsy source has only recently begun to be reported as a liquid biopsy source, with almost half of all publications on it being review articles. Experimental data revealed that cfDNA isolation protocols give a wide range of cfDNA yields, both from blood and seminal plasma. The addition of DTT to CKs has improved yields in seminal plasma and had a neutral/negative impact in blood plasma. Capillary electrophoresis and fluorometry reported much higher yields than PCR methods. While cfDNA yield and integrity were highly impacted, cfDNA methylation was not affected by isolation methodology or DTT. In conclusion, NucleoSnap was recognized as the kit with the best overall performance. DTT improved CK yields in seminal plasma. The in-house developed protocol has shown near-kit isolation performance. ddPCR LINE-1 assay for absolute detection of minute amounts of cfDNA was established and allowed for quantification of samples inhibited in qPCR. cfDNA methylation was recognized as a stable biomarker unimpacted by cfDNA isolation method. Finally, semen was found to be an abundant source of cfDNA offering potential research opportunities and benefits for cfDNA based biomarkers development related to male reproductive health.

MiR-182-5p and miR-375-3p Have Higher Performance Than PSA in Discriminating Prostate Cancer from Benign Prostate Hyperplasia.

Prostate cancer (PCa) is the most commonly diagnosed neoplasm among men. Since it often resembles benign prostate hyperplasia (BPH), biomarkers with a higher differential value than PSA are required. Epigenetic biomarkers in liquid biopsies, especially miRNA, could address this challenge. The absolute expression of miR-375-3p, miR-182-5p, miR-21-5p, and miR-148a-3p were quantified in blood plasma and seminal plasma of 65 PCa and 58 BPH patients by digital droplet PCR. The sensitivity and specificity of these microRNAs were determined using ROC curve analysis. The higher expression of miR-182-5p and miR-375-3p in the blood plasma of PCa patients was statistically significant as compared to BPH (p = 0.0363 and 0.0226, respectively). Their combination achieved a specificity of 90.2% for predicting positive or negative biopsy results, while PSA cut-off of 4 µg/L performed with only 1.7% specificity. In seminal plasma, miR-375-3p, miR-182-5p, and miR-21-5p showed a statistically significantly higher expression in PCa patients with PSA >10 µg/L compared to ones with PSA ≤10 µg/L. MiR-182-5p and miR-375-3p in blood plasma show higher performance than PSA in discriminating PCa from BPH. Seminal plasma requires further investigation as it represents an obvious source for PCa biomarker identification.

Comparison of diagnostic accuracy for eight SARS-CoV-2 serological assays.

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests have been suggested as an additional diagnostic tool in highly suspected cases with a negative molecular test and determination of seroprevalence in population. We compared the diagnostic performance of eight commercial serological assays for IgA, IgM, and IgG antibodies to the SARS-CoV-2 virus. MATERIALS AND METHODS: The comparison study was performed on a total of 76 serum samples: 30 SARS-CoV-2 polymerase chain reaction (PCR)-negative and 46 SARS-CoV-2 PCR-positive patients with asymptomatic to severe disease and symptoms duration from 3-30 days. The study included: three rapid lateral flow immunochromatographic assays (LFIC), two enzyme-linked immunosorbent assays (ELISA), and three chemiluminescence immunoassays (CLIA). RESULTS: Agreement between IgM assays were minimal to moderate (kappa 0.26 to 0.63) and for IgG moderate to excellent (kappa 0.72 to 0.92). Sensitivities improved with > 10 days of symptoms and were: 30% to 89% for IgM; 89% to 100% for IgG; 96% for IgA; 100% for IgA/IgM combination; 96% for total antibodies. Overall specificities were: 90% to 100% for IgM; 85% to 100% for IgG; 90% for IgA; 70% for IgA/IgM combination; 100% for total antibodies. Diagnostic accuracy for IgG ELISA and CIA assays were excellent (AUC ≥ 0.90), without significant difference. IgA showed significantly better diagnostic accuracy than IgM (P < 0.001). CONCLUSION: There is high variability between IgM assays independently of the assay format, while IgG assays showed moderate to perfect agreement. The appropriate time for testing is crucial for the proper immunity investigation.

Robert's Intragastric Alcohol-Induced Gastric Lesion Model as an Escalated General Peripheral and Central Syndrome, Counteracted by the Stable Gastric Pentadecapeptide BPC 157.

We redefined Robert's prototypical cytoprotection model, namely the intragastric administration of 96% alcohol in order to generate a general peripheral and central syndrome similar to that which occurs when major central or peripheral veins are occluded in animal models. With this redefinition, we used Robert's model to examine the cytoprotective effects of the stable gastric pentadecapeptide BPC 157. The intragastric administration of alcohol induced gastric lesions, intracranial (superior sagittal sinus) hypertension, severe brain swelling and lesions, portal and vena caval hypertension, aortal hypotension, severe thrombosis, inferior vena cava and superior mesenteric vein congestion, azygos vein failure (as a failed collateral pathway), electrocardiogram disturbances, and heart, lung, liver and kidney lesions. The use of BPC 157 therapy (10 µg/kg or 10 ng/kg given intraperitoneally 1 min after alcohol) counteracted these deficits rapidly. Specifically, BPC 157 reversed brain swelling and superior mesenteric vein and inferior vena caval congestion, and helped the azygos vein to recover, which improved the collateral blood flow pathway. Microscopically, BPC 157 counteracted brain (i.e., intracerebral hemorrhage with degenerative changes of cerebral and cerebellar neurons), heart (acute subendocardial infarct), lung (parenchymal hemorrhage), liver (congestion), kidney (congestion) and gastrointestinal (epithelium loss, hemorrhagic gastritis) lesions. In addition, this may have taken place along with the activation of specific molecular pathways. In conclusion, these findings clarify and extend the theory of cytoprotection, offer an approach to its practical application, and establish BPC 157 as a prospective cytoprotective treatment.

Prediction of schizophrenia using MAOA-uVNTR polymorphism: A case-control study.

CONTEXT: Schizophrenia has been associated with disorder of the dopamine system, which is downregulated by projections of the serotonin pathway. Dopamine and serotonin levels are regulated by a system of transporters and enzymes. In this research, dopamine transporter polymorphism (DAT-VNTR), serotonin transporter polymorphism (5-HTTLPR), monoamine oxidase A (MAOA-uVNTR), and catechol-o-methyl transferase (COMT Val158Met) polymorphisms have been investigated. AIMS: The aim of this study was to asses frequencies of these polymorphisms in the healthy control group and patients and to asses association with schizophrenia. SETTINGS AND DESIGN: Three hundred and fourteen healthy volunteers and 306 schizophrenia patients were included. Schizophrenia was diagnosed by Diagnostic and Statistical Manual-IV of the American Psychiatric Association, and mini international neuropsychiatric interview questionnaire was used for screening of healthy population. MATERIALS AND METHODS: Genotyping was performed using polymerase chain reaction (PCR) reaction followed by gel electrophoresis and PCR-restriction fragment length polymorphism. STATISTICAL ANALYSIS: Categorical data were analyzed using the Chi-square test, age between subgroups was compared using the Mann-Whitney test, and all polymorphisms were tested for Hardy-Weinberg equilibrium. Logistic regression analysis was used to set the prediction model of schizophrenia. RESULTS: Difference in genotype distribution was observed for COMT Val158Met in female and DAT-VNTR polymorphism in overall sample P = 0.021 and P = 0.028, respectively. Statistically significant association of MAOA-uVNTR and schizophrenia was observed after adjustment for anamnestic predictors of disease. P = 0.010, 80.45% participants were correctly classified. CONCLUSION: Our results suggest an association of MAOA-uVNTR polymorphism with schizophrenia. The difference in the distribution of COMT Val158Met and DAT-VNTR polymorphism support the involvement of dopamine system components in the pathogenesis of schizophrenia.