Immunoturbidimetric Method Research Articles

One-Component Dual-Readout Aggregation-Induced Emission Nanobeads for Qualitative and Quantitative Detection of C-Reactive Protein at the Point of Care.

Fluorescent lateral flow immunoassay (LFA) systems are versatile tools for sensitive and quantitative detection of disease markers at the point of care. However, traditional fluorescent nanoparticle-based lateral flow immunoassays are not visible under room light, necessitate an additional fluorescent reader, and lack flexibility for different application scenarios. Herein, we report a dual-readout LFA system for the rapid and sensitive detection of C-reactive protein (CRP) in clinical samples. The system relied on the aggregation-induced emission nanobeads (AIENBs) encapsulated with red AIE luminogen, which possesses both highly fluorescent and colorimetric properties. The AIENB-based LFA in the naked-eye mode was able to qualitatively detect CRP levels as low as 8.0 mg/L, while in the fluorescent mode, it was able to quantitatively measure high-sensitivity CRP (hs-CRP) with a limit of detection of 0.16 mg/L. The AIENB-based LFA system also showed a good correlation with the clinically used immunoturbidimetric method for CRP and hs-CRP detection in human plasma. This dual-modal AIENB-based LFA system offers the convenience of colorimetric testing and highly sensitive and quantitative detection of disease biomarkers and medical diagnostics in various scenarios.

Determination of cystatin C reference interval for children in Croatia.

Cystatin C is considered an early marker of kidney damage. The aim was to determine the reference interval in children since this information was not available from the test manufacturer. Included were children aged 0 to 18 years undergoing routine check without history of any renal disease. Cystatin C was measured by the immunoturbidimetric method, and creatinine by the enzymatic method on a Cobas c501 analyzer (Roche Diagnostics, Manheim, Germany). Reference intervals were determined according to the CLSI C28-A3 guidelines using a robust method and a nonparametric percentile method, depending on the sample size. The Schwartz's formula was applied to estimate glomerular filtration (eGFR) from cystatin C. The cystatin C reference interval for children aged 1-18 years (N = 204, median 8 years) was from 0.61 mg/L (90% CI: 0.53 to 0.64) to 1.08 mg/L (90% CI: 1.07 to 1.14). Differences according to sex were not found. For children aged 0-1 years (N = 29, median 5 months), the reference interval was from 0.60 mg/L (90% CI: 0.48 to 0.72) to 1.49 mg/L (90% CI: 1.36 to 1.61). The sample size was too small to test the difference according to sex. The eGFR was 76 (70-88) mL/min/1.73m2 for males and 83 (74-92) mL/min/1.73m2 for females. The cystatin C reference intervals for Croatian pediatric population according to age were determined. The cystatin C concentrations in children reach adulthood values after the first year. The cystatin C Schwartz's formula is applicable for eGFR calculation in children.

The Association of Serum Calprotectin with Fitness Indicators and Biochemical Markers in High-Level Athletes: A Continuous Dynamic Monitoring during One Competitive Season.

Twenty professional male water polo players (median age: 28 (22-42)) were included in this study. Serum creatine kinase activity was determined by the automated photometric UV method. The concentrations of calprotectin, C-reactive protein, and myoglobin were measured using an automated immunoturbidimetric method, while an automated immunochemistry method was employed for interleukin-6, troponin I, and cortisol determination. Tests of repeated strength, maximal strength, and static strength were used to evaluate physical activity. Serum calprotectin concentrations expressed in median and IQR were significantly different: T1: 2.92 g/mL (2.47; 3.86); T2: 2.35 g/mL (1.26; 2.87); T3: 2.27 g/mL (1.60; 3.27); and T4: 1.47 g/mL (1.04; 2.85) (p = 0.004). Cortisol concentration and CK activity showed significant changes among phases (p = 0.049 and p = 0.014, respectively). Each physical activity examined showed a significant seasonal decrease (all p values were 0.001). Calprotectin serum concentration and indicators of muscular injury, inflammation, and physical activity were found to be correlated during particular stages of the seasonal examination. Calprotectin values determined throughout one competitive season decreased as training intensity among water polo players increased. Serum calprotectin concentrations and indicators were related to biochemical markers of inflammation and muscle damage.

Variation in Hemoglobin A1c Linked to Hemoglobin S: Comparison of Analysis Results from ion Exchange Chromatography and an Immunoturbidimetric Method

Hemoglobin S can interfere with the measurement of glycated hemoglobin, an essential tool for diagnosing and monitoring diabetes. The objective of this study is to evaluate the analytical performance of 2 glycated haemoglobin assay methods.
 A prospective cross-sectional study was conducted where 186 patients (61 homozygous sickle cell disease, 61 AA type subjects and 64 AS type subjects) were recruited. Glycated hemoglobin was measured by immunoturbidimetry method and ion exchange chromatography.
 The coefficient of variation (CV) of the repeatability is 2.34% and 1.13% (normal rate) ; 2.32% and 1.65% (high rate) respectively for the immunoturbidimetric method and ion exchange chromatography. In reproducibility, the CV obtained are 3.23% and 2.64% (normal rate) and 3.27% and 2.22% (high rate), respectively for the immunoturbidimetric method and the ion exchange chromatography. Linearity is satisfactory for both methods. Mean glycated hemoglobin values ​​show no significant difference (the P-value is equal to 0.09, 0.17 and 0.70 respectively in subjects AA, AS and SS) in the 2 methods for patients with the same hemoglobin electrophoretic profile.
 The analytical performances of the 2 methods are good but their use is not recommended in the biological diagnosis of diabetes and pre-diabetes due to interference from hemoglobin S, especially in the case of homozygous sickle cell disease or in the case of composite heterozygosity.

The correlation between Plasma Fibrinogen Levels and Glycemic Status in type 2 Diabetes Mellitus

Abstract Introduction/Objective Background: Fibrinogen is considered a well-known contributor to the progression of atherosclerotic lesions and a predictor of cardiovascular events. Patients with diabetes mellitus (DM) have higher levels of fibrinogen, which may raise their thromboembolic risk for cardiovascular disease (CVD). Plasma fibrinogen is an indicator of inflammation, and endothelial dysfunction has been described as an independent risk factor for cardiovascular morbidity in type-2 diabetes mellitus (T2DM) patients. Poor glycemic control has been reported to be associated with hyperfibrinogenemia. Aim This study aims to estimate the levels of fibrinogen in patients with type-2 diabetes mellitus and determine its correlation with glycated hemoglobin levels. Methods/Case Report Subjects and methods: It is a cross-sectional analytic study conducted in the Benghazi Diabetic Clinic, Libya. Patients who were diagnosed with type 2 diabetes mellitus, either newly detected or already on diet or treatments, were included in the study during the period between May 2022 to December 2022. A total of 231 adult subjects were selected, with ages ranging from 20-80 years. Random blood sugar levels were assessed. Plasma fibrinogen levels (mg/dl) were estimated by the "Clauss method" and glycosylated hemoglobin (%) by the "immunoturbidimetric method." Patients were categorized into three groups depending upon the HbA1c values, i.e., into <5.7% (good control), 5.7-6.5% (fair control), and >6.5% (poor control). Results (if a Case Study enter NA) Result: Out of 231 subjects, (40.6%) were males and (59.4%) were females, with a mean±SD of age was (47.03±15.54) years. There was a significant positive correlation between fibrinogen and HbA1c in the fair control group (r=0.424, p=0.003) and the poor control group (r=0.369, P=0.000). Moreover, the difference between fibrinogen and age in the good control group was significant (p>0.05), whereas no significance was recognized in other groups. Conclusions Plasma fibrinogen levels are elevated in type 2 DM, and this elevation was associated with poor glycemic control; increased HbA1c levels. The other parameters which correlated with plasma fibrinogen levels were age. Lowering plasma fibrinogen levels could be an integral approach to cardiovascular complications reduction in type-2 DM.

Assessment of urinary podocalyxin as a biomarker of early diagnosis of hypertensive nephropathy

Chronically high blood pressure-related kidney injury is known as hypertensive nephropathy (HN). Podocyte damage in the pathogenesis of this disease can result in the release of the sialoglycoprotein podocalyxin into the urine, so podocalyxin may be useful in the early diagnosis of HN. The purpose of the study was to examine the relationships between urine podocalyxin level and clinical and biochemical parameters in individuals with HN and to assess the diagnostic utility of urinary podocalyxin as an early marker of HN. Participants (114 individuals) were enrolled in this cross-sectional study, including 30 healthy controls and 84 patients with clinically proven chronic hypertension (CH). Biochemical tests were performed on the blood samples. Urinary microalbumin and creatinine levels were measured using immunoturbidimetric and spectrophotometric methods, respectively; urinary podocalyxin level was estimated with ELISA. All CH patients were classified into subgroups according to urine microalbumin/creatinine ratio (UM/CR) and the stage of chronic kidney disease (CKD). The results obtained showed that urinary podocalyxin level was significantly increased in both UM/CR and CKD staging subgroups compared with the healthy control group. A gradual increase in urinary podocalyxin level with CKD stage, especially in IV and V stages, and the higher sensitivi­ty of urinary podocalyxin as compared to UM/CR ratio in early detection of HN was demonstrated. It was concluded that urinary podocalyxin may be an important and highly sensitive marker for early diagnosis of hypertensive nephropathy in patients with chronic hypertension. Keywords: creatinine, diagnostic marker, hypertensive nephropathy, microalbumin, podocalyxin, urine

Association of Cathepsin K and Tartrate- Resistant Acid Phosphatase-5b in Different Stages of Rheumatoid Arthritis Patients in South Indian Population

The aim of the study was to identify effective biomarkers of changes in bone mineral density (BMD) at different stages of rheumatoid arthritis patients from South Indian population, using disease activity score in 28 joints (DAS28) which is being used as a measurement for assessing disease activity in patients with rheumatoid arthritis. The study was carried out in 92 rheumatoid arthritis patients. Serum level of tartrateresistant acid phosphatase-5b (TRAP-5b) and cathepsin K was measured using ELISA. Serum rheumatoid factor (RF) and C-reactive protein (CRP) was recorded by the attending rheumatologists. The C-reactive protein (CRP) was quantified using a latex immunoturbidimetric method. The rheumatoid arthritis (RA) was measured by turbidimetric immunoassay method. The bone mineral density (BMD) T-score was calculated according to WHO guidelines. Reverse transcription PCR (RT-PCR) was used to determine the expression of cathepsin K. The rheumatoid arthritis (RA) patients were categorized into three groups based on the DAS28 score as inactive (DAS ≤ 3.2), moderately active (DAS > 3.2 ≤ 5.1) and very active (DAS > 5.1) at the time of admission. Out of 92 patients, 16 (17.4%) patients had inactive disease condition, 42 (45.6%) patients had moderately active disease condition and 34 (37%) patients had very active disease condition. The mean BMD was significantly lower in very active (0.28 ± 0.04; p < 0.001) as compared with moderately active (0.71 ± 0.13; p < 0.001) and inactive (1.21 ± 0.14). TRAP5b and cathepsin K showed significant increases in very active group (p < 0.001) as compared with moderately active (p < 0.001) and inactive groups. In conclusion, The biomarker TRAP-5b and cathepsin K identified in this study may become a new and highly specific biomarker for rheumatoid arthritis

Validation of the fCAL turbo immunoturbidimetric assay for measurement of calprotectin in porcine and bovine fecal samples

The concentration of calprotectin in feces is a well-studied marker of gastrointestinal inflammation in humans. However, little is known about fecal calprotectin in farm animals. In this work, we have validated an immunoturbidimetric method for fecal calprotectin (Bühlmann fCAL® turbo assay, Schönenbuch, Switzerland) in porcine and bovine fecal samples. Linearity was evaluated by serial dilution (R2 > 0.97 was obtained for both species). Accuracy was assessed by a recovery study, with results between 80 and 120% for low, medium, and high samples in both species. Intra- and inter-assay variability was <20%. Limit of detection was 6.4 μg/g in pig and 5.3 μg/g in cow. Limit of quantification was 13.4 μg/g (pig) and 11.1 μg/g (cow). Additionally, clinical validation has been included to evaluate the ability of the assay to detect inflammatory status in the intestine under different management conditions. In experiments with porcine, it was found that piglets treated with ZnO had lower concentrations of fecal calprotectin. In a second experiment in bovine, calves with diarrhea had higher concentration of fecal calprotectin.The Bühlmann fCAL® turbo assay is suitable for measurement of calprotectin in porcine and bovine fecal samples. Moreover, fecal calprotectin could be a good biomarker of intestinal inflammation in both species.

A-118 Evaluation of Mindray Cystatin C Assay

Abstract Background Cystatin C is a low molecular weight non-glycosylated alkaline protein, with a molecular weight of 13kDa. It is formed by all nucleated cells in human at a constant rate. The only pathway of Cystatin C in vivo is excretion through glomerular filtration and absorbed by proximal renal tubular epithelial cells, which was decomposed finally. Therefore, the concentration of Cystatin C in serum is almost not affected by external factors, such as weight, age, gender, etc. Compared with creatinine and urea, it is an ideal endogenous marker for monitoring glomerular filtration function. Currently, the most methods on the market for testing Cystatin C are either latex enhanced immunoturbidimetric method, or immunoturbidimetric method. These methods usually use polyclonal antibodies from different animal species, which are sometime susceptible to the interference from rheumatoid factors reported in the literature, resulting in false positive results. The anti-RF interference ability of this reagent was enhanced by selecting the optimal antibody and optimizing reagent formulation. This study evaluated performances of the Cystatin C assay on Mindray BS-2000M system. Method Mindray Cystatin C assay is based on latex immunno-agglutination. Latex nanoparticles were coated with anti-Cystatin C antibody. Reagent of this assay contains R1 buffer and R2 with latex particles coupled with anti-Cystatin C antibodies. The assay starts with R1 and sample incubation for 5 min and then R2 is added. After another 5 min of incubation, agglutination occurs, leading to turbidity increasing. The turbidity is measured at 570 nm. Multi-level (5-point) calibration is used. Cystatin C concentrations are obtained from the calibration curve with signals from the assay with patient samples. Following basic assay performances were evaluated: precision, limit of detection, linearity, method comparison, interference and high dose hook. Results The precision study followed EP05-03 protocol. The repeatability and within-lab CVs on the BS-2000M system with 2 QC and 3 patient samples were ranged from 0.9% to 5.3%, respectively. Limit of detection was 0.1 mg/L and linearity was given up to 8.63 mg/L. The assay (y) shows good accuracy when compared with Roche Cystatin C (x) assay on the cobas 701: y = 1.0055x - 0.0268 (r2 = 0.9994, n = 109). The assay showed no significant with bilirubin up to 40 mg/dL, with hemoglobin up to 500 mg/dL, with lipids up to 500 mg/dL, and with rheumatoid factors up to 1200 IU/mL that is about 0.3% out of a total of 55 184 samples collected from our customers in China with RF measurements. No prozone was observed with the assay up to the high cystatin C concentration of 800 mg/L tested. Conclusion From this evaluation, we confirmed that Mindray Cystatin C assay can measure serum Cystatin C precisely and accurately, and with good performance against rheumatoid factors interference. It is suitable for use in routine clinical laboratories.

A-210 Comparison of Two Lipoprotein(a) Methods: Validation, Sample Stability, and Patient Characteristics

Abstract Background Lipoprotein(a) [Lp(a)] is now thought of as an independent risk factor for coronary heart disease, stroke, and calcific aortic valve stenosis, and drugs specifically targeting Lp(a) are showing promise in clinical trials. Our laboratory recently validated two commercially available methods and used them to characterize a randomly selected group of samples from an outpatient clinic in the United States. Since the assays have different stated sample stabilities, this was also compared. Methods Both the Roche Lp(a) and the Randox Lp(a) immunoturbidimetric methods were run on a Roche cobas c501 platform across several months. In the U.S., the Roche assay is calibrated in mg/dL, while Randox provides both mg/dL and nmol/L units. Assays were tested for reproducibility, intermediate precision and accuracy. Stability of samples and AMR were established. Additionally, 78 residual samples were randomly selected to assess the percentage with high Lp(a) in our patient population. Results Method validation showed acceptable within-run and between-run precision as both assays met or exceeded manufacturer’s specifications. The stated AMR for the Roche assay is 6.0 – 80.0 mg/dL; linearity was verified through 90.0 mg/dL using the Randox calibrators. For the Randox assay, linearity was verified for the manufacturer’s AMR of 5.2–206.0 nmol/L using estimated values assigned to the Roche calibrators. Both assays allow for x3 dilutions to extend the range. Method comparison of the Randox results (nmol/L) was performed with samples sent to Quest. Deming regression results were acceptable (r = 0.995, slope 1.2, y-intercept—2.3). The stated sample stability from Roche is 8 h at room temperature and 48 h at 2–8oC; however, our results suggest 7 h at room temperature and up to 10 days at 2–8oC. Our study also extended the number of freeze-thaws to two. The Randox package insert gives no information on freeze-thaws or room temperature stability, but states stability at 4oC for 14 days. In our assessment, some samples were not stable past 10 days at 2–8oC. We found samples stable for 2 days at room temperature and up to one month at −20oC. Three freeze-thaws was acceptable. Our patient population included 49 Caucasians, 19 Blacks, 2 Asians, 3 Hispanics, and 5 with race unknown. The median Lp(a) for Blacks was 151.1 nmol/L (65.1 mg/dL) while for Caucasians it was 17.9 nmol/L (14.5 mg/dL) for samples within range. The extended range with dilutions allowed all high values to be quantified; however, 12 were below the AMR for the Roche method and 8 below the AMR for Randox method. Based on the Roche assay, 20 (25.6%) subjects had Lp(a) &amp;gt; 50 mg/dL and would be considered to have increased cardiovascular risk. By the Randox assay, the same 20 had an Lp(a) &amp;gt; 100 nmol/L, while 19 had Lp(a) &amp;gt; 125 nmol/L. Conclusions Both assays met performance goals and showed no significant difference in % subjects with elevated risk for cardiovascular events using typical cut-offs. Our data suggests greater stability of samples at room temperature and additional stability to freeze-thaws when using the Randox assay.

ANALYSIS OF URINE MICROALBUMIN LEVELS ON SERUM CREATININE IN PATIENTS WITH DIABETES MELLITUS IN THE CLINICAL LABORATORY OF MEDAN PRODIA

Diabetes Mellitus is a degenerative disease that requires regular control. This disease occurs due to the pancreas not being able to produce enough insulin and/or when the body cannot produce insulin effectively so that it can cause various complications in the patient's organs. The purpose of this study was to determine the levels of Urine Microalbumin and Serum Creatinine at Prodia Clinical Laboratory Jl. Lt. Gen. S. Parman No. 17/223 G Medan. This research was conducted from August to October 2022 at the Clinical Laboratory of Prodia Medan. This type of research is descriptive Cross Sectional. This examination uses Immunoturbidimetric and Enzymatic Colorimetric methods with a total sample of 100 samples or the total of the population. The results of the research conducted showed that the number of Diabetes Mellitus patients at the Prodia Clinical Laboratory in Medan with gender as many as 48 men (48%) and 52 women (52%) samples. Normal Urine Microalbumin levels were 30 samples (30%) and High Urine Microalbumin levels were 70 samples (70%). Normal creatinine levels were 47 samples (47%) and abnormal creatinine levels were 53 samples (53%).

Plasma zinc, copper and serum ceruloplasmin levels of autism spectrum disorder children in Bangladesh

Neural and cognitive processes require zinc and copper homeostasis and a normal zinc/copper ratio. Ceruloplasmin, an intrinsic antioxidant protein, maintains copper homeostasis, which might also influence autism spectrum disorder (ASD). ASD children are frequently reported with altered levels of these elements with wide geographical variations. This study evaluated any alteration in plasma zinc, copper, zinc/copper ratio and serum ceruloplasmin levels in Bangladeshi ASD children with respect to healthy controls. A cross-sectional study was conducted on 67 children aged 2 to 9 years of both sexes. Among them, 35 had ASD, while 32 were age, sex and body mass index (BMI) matched apparently healthy children. Plasma zinc and copper levels were estimated by the flame atomic absorption spectrophotometry method. Serum ceruloplasmin levels were estimated by the immunoturbidimetric method. Zinc and zinc/copper ratio in the 2–9 years old ASD children group were significantly lower (p=0.032 and p=0.002 respectively). On the other hand, copper (p=0.020) and ceruloplasmin (p = 0.045) levels were significantly higher than those of apparently healthy children. ASD was significantly associated with zinc deficiency (p=0.000) and copper toxicity (p=0.05). All children were again divided into 2–5 and 6–9 years age groups according to laboratory reference values for zinc and copper. Copper toxicity was significantly associated with ASD in the 2–5 years old age group (p=0.011), with a significant difference in plasma copper levels (p=0.009) and zinc/copper ratio (p=0.001) but not serum ceruloplasmin levels (p=0.110) compared to healthy controls. Serum ceruloplasmin was positively associated with plasma copper in ASD children of all age groups. This study shows that ASD in Bangladesh can be associated with low plasma zinc and high plasma copper and serum ceruloplasmin levels.

Implementation of pro- and anti-inflammatory mechanisms of sirtuin-1 in patients with chronic obstructive pulmonary disease and chronic coronary syndrome and their combination

One of the key pathogenetic aspects of chronic obstructive pulmonary disease (COPD) and chronic coronary syndrome (CCS) is endothelial dysfunction. Further study on this pathological process is relevant for a better understanding of the course and increasing the treatment effectiveness of these diseases. In this context, in recent years, there has been a growing body of evidence about sirtuins which, according to the latest research results, were capable of rebalancing vasoconstrictory and vasodilatory mediators, regulating the balance in the protease-antiprotease system, protecting endothelial cells from oxidative stress and may be useful in relation to the pathogenesis of COPD and CCS. Aim. To study changes in the levels of serum endothelial nitric oxide synthase (NOS3/eNOS) and sirtuin 1 (SIRT1) in patients with a combined course of COPD and CCS and with COPD or CCS alone. Materials and methods. In order to study endothelial dysfunction and the role of SIRT1 in the processes of its development, we examined 60 patients with combined course of COPD and with CCS or COPD alone. The control group included 10 apparently healthy individuals. Levels of SIRT1 and NOS3/eNOS were studied by enzyme-linked immunosorbent assay, and high-sensitivity C-reactive protein (hsCRP) – by immunoturbidimetric method; clinical data and medical records were analyzed. Commonplace statistical analyses were used. Results. The results have shown that the serum level of SIRT1 in patients with COPD and CCS was lower than that in the control group. The level of hsCRP was significantly higher in patients with the studied pathology than that in apparently healthy individuals and was the highest in patients with combined COPD and CCS. The study on serum NOS3/eNOS has showed the highest concentration in COPD patients, and it was significantly higher in all groups of patients with the studied pathology than that in healthy individuals. A direct correlation has been found between the serum level of SIRT1 and hsCRP and an inverse correlation – with the level of NOS3/eNOS. Conclusions. Thus, the pleiotropic effects and multifaceted molecular interactions of SIRT1 are a promising direction in the search for effective new therapeutic strategies in COPD and CCS.

The Significance of CXCL1 and CXCR1 as Potential Biomarkers of Colorectal Cancer.

The CXCL1/CXCR2 and CXCL8-CXCR1/CXCR2 axes are under intensive investigation as they appear to regulate the progression and invasion of colorectal cancer (CRC). Growing evidence demonstrates the elevated expression of these proteins in CRC. However, a majority of relevant studies have been performed on CRC tissues using immunohistochemical techniques. Our study is the first to evaluate the diagnostic significance of serum CXCL1 and CXCR1 levels in CRC patients in comparison to well-established tumor markers, such as the carcinoembryonic antigen (CEA), and markers of inflammation, such as C-reactive protein (CRP). Thus, the aim of our study was to assess whether circulating serum levels of CXCL1 and CXCR1 might be candidates for novel biomarkers in the diagnosis and progression of CRC. The study was performed on 76 subjects, including patients with CRC and healthy volunteers as a control group. Serum concentrations of CXCL1, CXCR1, and the classical tumor marker (CEA) were measured using immunoenzyme assays, while CRP levels were assessed with the immunoturbidimetric method. Serum CXCL1 levels were statistically significantly increased in CRC patients when compared to healthy subjects, and similar results were found for CEA and CRP levels. The percentage of elevated concentrations of CXCL1 and CXCR1 was higher than that of the classical tumor biomarker and increased in the combined measurement of these proteins with CEA. In addition, among all proteins tested, serum CXCL1 seems to be the best indicator in the differentiation between CRC patients with nodal involvement and patients without the presence of lymph node metastasis. Our preliminary results indicate the role of serum CXCL1 and CXCR1 in the diagnosis of CRC, particularly in the combined measurement with CEA.

Post-Traumatic Stress Response and Appendicitis in Children-Clinical Usefulness of Selected Biomarkers.

Acute appendicitis is an inflammatory process which is one of the most frequent global causes of surgical interventions in children. The goal of the study was to determine whether acute phase proteins, that is, C-reactive protein (CRP), procalcitonin (PCT) and neutrophil gelatinase-associated lipocalin (NGAL), interleukin 6 (IL-6), transforming growth factor-beta1 (TGF-β1) and cortisol (HC) play a role in the pathomechanism of post-trauma stress response of the organism and to establish the impact of the applied surgical procedure and/or of inflammation on their concentrations. An additional purpose was to establish the clinical usefulness of the studied biomarkers in the diagnostics of appendicitis. CRP concentrations were quantified via the immunoturbidimetric method, while the levels of IL-6 and PCT were assessed using a bead-based multiplexed immunoassay system in a microplate format (Luminex xMAP technology); NGAL, TGF-β1 and cortisol concentrations were determined via the enzyme-linked immunosorbent assay (ELISA) technique. All the investigated biomarkers were assayed twice, i.e., immediately before the surgery and 12-24 h after its completion. Significant increases in CRP, IL-6 and PCT concentrations were found in all children subjected to laparoscopic surgeries (p = 0.001, p = 0.006, and p = 0.009, respectively) and open (classic) surgeries (p = 0.001, p = 0.016, and p = 0.044, respectively) compared to the initial concentrations. The patients undergoing classical surgery moreover presented with significant (p = 0.002, and p = 0.022, respectively) increases in NGAL and TGF-β1 levels after the procedures. In a group of children undergoing laparoscopic surgery, the appendicitis induced an increase in cortisol concentration, whereas in patients undergoing classical surgery the increase in the levels of this biomarker was caused by the type of performed surgical procedure. Simultaneously assaying the levels of CRP, NGAL and IL-6 (p = 0.008, p = 0.022, and p = 0.000, respectively) may prove useful in clinical practice, enabling the diagnosis of appendicitis in paediatric patients reporting to a hospital with abdominal pains, in addition to data from anamnesis and from clinical or ultrasound examination. The performed study confirms the participation of examined biomarkers in the pathomechanism of post-injury stress reaction of the organism to surgical trauma.

Lipoprotein (a) as a marker of hereditary lipid metabolism disorders in patients with early manifestation of coronary artery disease

Background: The clinical phenotype of familial hypercholesterolemia (FH) combined with the classical genetic defects of this disease increase the risk of coronary artery disease (CAD) in the case of a high level of lipoprotein (a) (Lp(a)). Aim: To assess the association of hereditary disorders of lipid metabolism with a high level of Lp(a) in the case of an early manifestation of CAD in the absence of the FH phenotype. Methods: We studied 81 patients with premature CAD (at the age up to 55 years in men and up to 60 years in women). Lp(a) was measured in the blood serum by the immunoturbidimetric method. We performed the molecular genetic testing, using massively parallel sequencing, which included the following panel of genes: ABCA1, ABCG1, ABCG5, ABCG8, ANGPTL3, APOA1, APOA2, APOA4, APOA5, APOB, APOC1, APOC2, APOC3, APOE, APOH, CETP, CH25H, CIDEC, CREB3L3, GPD1, GPIHBP1, LCAT, LDLR, LDLRAP1, LIPA, LIPC, LIPE, LIPG, LMF1, LPA, LPL, MTTP, MYLIP, NPC1, NPC1L1, NPC2, PCSK9, PLTP, PPP1R17, SAR1B, SCARB1, STAP1. Results: 24 (29.6%) patients had Lp(a) 30 mg/dl and were more likely to have a family history of CAD (70.8% vs 42.1%, p=0.05). In patients with a confirmed FH phenotype, there were no pathogenic variants associated with hereditary disorders of lipid metabolism. 4 patients with Lp(a) 30 mg/dl without a confirmed FH phenotype appeared to be carriers of pathogenic variants in the genes of lipid metabolism (LDLR p.Glu763Asp, ABCA1 p.Arg1128His, APOA5 p.His321Le), as well as of a not previously registered variant in the LIPE gene NM_005357.4:c. 2312TC. Conclusions: Lp(a) can be an appropriate marker for revealing pathogenic variants in the genes of the lipid metabolism system in patients without the clinical FH phenotype with an early CAD manifestation.

Effect of Probiotic Therapy on the Vagina Microbiota and the Humoral Link of Immunity in Bacterial Vaginosis

The aim of the research is to determine the effect of the probiotic preparation «Dialak» (dietary supplement), which includes the strain Lactobacillus casei IMV B-7280, on the vaginal microbiota and humoral immunity in women with bacterial vaginosis (BV). Methods. 40 female patients aged 20—45 years with disturbed vaginal microbiota and 10 healthy individuals were examined. The verification of 3 types of vaginal biocenosis states, namely normocenosis, intermediate type, and vaginal dysbiosis, was carried out on the basis of the Recommendations for the Treatment of Sexually Transmitted Infections Weekly Morbidity and Mortality Report (2021) and laboratory diagnostic methods according to the well-known criteria proposed by R. Amsel. Female patients with an intermediate type of BV (group 1) received suppositories and capsules of the probiotic (once daily) for 10 days. Women with vaginal dysbiosis (group 2) received metronidazole in a dosage of 500 mg twice a day for 7 days during the first stage, and then 1 suppository at night and oral capsules of the probiotic in the morning for 10 days during the second stage. The studied vaginal secretion was stained by the Gram method in the Kopeloff modification and also sown on nutrient media to determine facultatively anaerobic and obligately anaerobic microorganisms. Microorganism identification was carried out on the basis of morphological, cultural, biochemical, and antigenic properties according to the classification of D. H. Bergey (2009). The activity of humoral immunity was determined by evaluating the number of B-lymphocytes in the peripheral blood of patients using flow cytometry, as well as the levels of serum Ig A, M, and G before treatment and aft er 1 month using the immunoturbidimetric method and the Cobas 6000 test system from Roche Diagnostics (Switzerland). Results. When analyzing the vaginal microbiota in two groups of patients before treatment, a decrease in the number of Lactobacillus spp. and Bifidobacterium spp. and a significant increase in the number of obligate anaerobic microorganisms, including Gardnerella vaginalis, were found compared to the control group. Before treatment, the number of Lactobacillus spp. in women of group 2 was lower compared to group 1. In patients with vaginal dysbiosis before treatment, the number of obligate anaerobic microorganisms was higher than in patients with bacterial vaginosis, except for Eubacterium spp. At the same time, in women in both comparison groups, the indicators of the humoral immune response were partially disrupted, as evidenced by a decrease in the level of IgG and IgA (in women of group 2) in the serum against the normal level of B lymphocytes (CD19+ cells). However, these patients showed an increase in the IgM level in the serum, which may be due to the development of anaerobic microflora. After treatment, the number of Lactobacillus spp. and Bifidobacterium spp. in the vagina of women in both comparison groups increased compared to the indicators before treatment. However, the number of these bacteria in the vagina of patients with dysbiosis remained lower compared to patients with BV. In both groups, normalization of the number of obligate anaerobic microorganisms, including G. vaginalis, except for Veillonella spp., was also observed. After treatment, the humoral immune response indicators were normalized as well: the level of serum IgG and IgA increased, and the content of serum IgM decreased. Conclusions. In BV patients, the probiotic «Dialak» normalizes the vaginal microbiota, which was confirmed by increasing the number of Lactobacillus spp. and Bifidobacterium spp. along with decreasing the number of anaerobic microorganisms, including G. vaginalis, against the background of the dynamic disappearance of clinical signs of the disease, as well as restoration to the normal level of indicators of the immunity humoral link. The obtained data indicate the effective therapeutic effect of the probiotic «Dialak» on BV.

Molecularly imprinted polymer dual electrochemical sensor for the one-step determination of albuminuria to creatinine ratio (ACR)

The urinary albumin to creatinine ratio (ACR) is a convenient and accurate biomarker of chronic kidney disease (CKD). An electrochemical sensor for the quantification of ACR was developed based on a dual screen-printed carbon electrode (SPdCE). The SPdCE was modified with carboxylated multiwalled carbon nanotubes (f-MWCNTs) and redox probes of polymethylene blue (PMB) for creatinine and ferrocene (Fc) for albumin. The modified working electrodes were then molecularly imprinted with coated with polymerized poly-o-phenylenediamine (PoPD) to form surfaces that could be separately imprinted with creatinine and albumin template molecules. The seeded polymer layers were polymerized with a second coating of PoPD and the templates were removed to form two different molecularly imprinted polymer (MIP) layers. The dual sensor presented recognition sites for creatinine and albumin on different working electrodes, enabling the measurement of each analyte in one potential scan of square wave voltammetry (SWV). The proposed sensor produced linear ranges of 5.0–100 ng mL−1 and 100–2500 ng mL−1 for creatinine, and 5.0–100 ng mL−1 for albumin. LODs were 1.5 ± 0.2 ng mL−1 and 1.5 ± 0.3 ng mL−1, respectively. The dual MIP sensor was highly selective and stable for seven weeks at room temperature. The ACRs obtained using the proposed sensor compared well (P > 0.05) with the results from immunoturbidimetric and enzymatic methods.

Correlation Between IL-8, C-Reactive Proteins (CRP) and Neutrophil to Lymphocyte Ratio (NLR) as Predictor of Mortality in COVID-19 Patients with Diabetes Mellitus Comorbidity.

COVID-19 is caused by SARS-CoV-2 and has a wide range of symptoms. While Diabetes Mellitus (DM) is a common comorbidity in COVID-19 patients, it is the main comorbidity in non-surviving COVID-19 patients. Interleukin-8 (IL-8) is a cytokine that has been correlated with severity and mortality in COVID-19 patients, but its role in COVID-19 patients with DM comorbidity and its relationship with NLR and CRP as markers of inflammation are not yet fully understood. To investigate the correlation between IL-8, NLR, and CRP in COVID-19 patients with DM comorbidity. A cross-sectional study was conducted at the Integrated Infectious Disease Installation of Dr. Saiful Anwar Malang Hospital from June to November 2021 using consecutive sampling. IL-8 was measured using the ELISA method with Legendmax® Human IL-8. NLR was measured using flow cytometry, while CRP was measured using the immunoturbidimetric method with Cobas C6000®. Patient outcomes were obtained from medical records. A total of 124 research subjects participated in the study. IL-8 and CRP levels were significantly higher (p < 0.05) in COVID-19 patients with DM comorbidity, and were also significantly higher (p < 0.05) in non-surviving COVID-19 patients. Overall, there was a positive correlation between IL-8 and CRP (r = 0.58, p < 0.05). There was also a positive correlation between IL-8 (r = 0.58; p < 0.05), NLR (r = 0.45, p < 0.05), CRP (r = 0.54, p < 0.05) and mortality in COVID-19 patients with DM comorbidity. The presence of DM comorbidity increased IL-8 levels and aggravated inflammation in COVID-19 patients, thereby increasing the risk of mortality. IL-8, CRP and NLR levels were higher in non-surviving COVID-19 patients with DM comorbidity, indicating that they could serve as good predictors of poor outcomes in this patient population.

Analysıs of Urınary Albumın and Urınary Synaptopodın Levels in Type 2 Dıabetes Mellıtus Subjects

Hyperglycemia from diabetes mellitus affects many body organs and interferes with normal function. Kidney function decreased in approximately one-third of patients with diabetes mellitus before the development of albuminuria. The purpose of this study was to analyze differences in urine albumin and urine synaptopodin levels in type 2 diabetes mellitus (T2DM) and non-DM subjects, differences in urine synaptopodin levels in T2DM subjects with and without nephropathy, to analyze the correlation between urine albumin and urine synaptopodin in T2DM subjects and the cut-off analysis of sensitivity and specificity of urinary synaptopodin in diagnosing diabetic nephropathy. A sample of 60 subjects comprised 40 T2DM subjects and 20 non-DM subjects. Urinary synaptopodin levels were examined using the ELISA method, and albuminuria levels using the immunoturbidimetric method. Based on statistical analysis, the results showed that there were differences in urine albumin levels in T2DM and non-DM subjects (p*= &lt; 0.001), there were differences in urine synaptopodin levels in T2DM and non-DM subjects (p*= &lt; 0.001), there were no differences in urine synaptopodin levels with and without nephropathy in T2DM subjects (p*= 0.090), a relationship was found between urine albumin and urinary synaptopodin in T2DM subjects (p*= 0.048, r= 0.314) and the cut off of urinary synaptopodin in diagnosing nephropathy was ³0.39 ng/mL, sensitivity 64.7% and specificity 56.5%. We recommend further prospective studies with larger sample sizes to compare urinary synaptopodin levels and microalbuminuria (MAU) as markers for early detection of DN in T2DM subjects.