Biochemia medica最新文献

Introduction: Adrenocorticotropic hormone (ACTH) is a peptide secreted by pituitary gland that plays an important role in regulating cortisol secretion. Its determination is difficult because of instability in whole blood. Several factors that influence ACTH stability in blood before analysis have been identified: temperature, hemolysis, time to centrifugation and presence of protease inhibitors. Published results on ACTH whole blood stability seem contradictory.

Materials and methods: We performed a stability study in 10 healthy volunteers. Three different conditions were tested: ethylenediaminetetraacetic acid (EDTA) at 4 °C, EDTA + aprotinin at 4 °C, EDTA + aprotinin at room temperature. Stability was evaluated for 8 hours. Adrenocorticotropic hormone measurements and hemolysis index were performed respectively on Cobas e602 and c701 (Roche Diagnostics, Mannheim, Germany). We compared percentage deviations with total change limit using a threshold of 7.5%.

Results: We showed that ACTH is stable 8 hours with EDTA at 4 °C, 4 hours with EDTA + aprotinin at 4 °C and 2 hours with EDTA + aprotinin at 22 °C.

Conclusions: Aprotinin does not appear to give ACTH greater stability but can be used without exceeding 4 hours at 4 °C. Refrigerated pouch transport also seems to be more appropriate for ACTH in whole blood.

Structural and functional alterations in platelets are an actual problem that requires more attention. The treatment of these illnesses proves challenging, inefficient and heavily relies on platelet donations. A difficult task confronting science is producing platelets in vitro, which calls for meticulous examination of factors affecting platelet generation. It is known that megakaryocytes produce platelets in vitro and in vivo differently: in the laboratory we can get a smaller number of platelets compared to the human body. This review primarily examines the stages of megakaryocyte maturation and the processes involved in platelet formation. The article reflects the results of both fundamental research on the problem and the new results obtained over the past decade. Currently, most scientists accept the pro-platelets theory of platelet formation. This review aims to explore in detail each stage of pro-platelet formation and the platelet formation process. It explains on the processes of polyploidization, endomitosis, and apoptosis, as well as the functions of structural cell components (microtubules, mitochondria, T- and α-granules) and pro-platelet migration. The microenvironment influence is acknowledged for the osteoblastic and vascular niches that affect thrombocytopoiesis. The additional aspect is the contribution of specific proteins to thrombocytopoiesis such as RhoA, β1-tubulin, cytokines IL-6, IL-8, Toll-like receptors, etc.

The investigation of biomarkers is constantly evolving. New molecules and molecular assemblies, such as soluble and particulate complexes, emerged as biomarkers from basic research and investigation of different proteomes, genomes, and glycomes. Extracellular vesicles (EVs), and glycans, complex carbohydrates are ubiquitous in nature. The composition and structure of both reflect physiological state of paternal cells and are strikingly changed in diseases. The EV-associated glycans, alone or in combination with soluble glycans in related biological fluids, used as analytes, aim to capture full complex biomarker picture, enabling its use in different clinical settings. Bringing together EVs and glycans can help to extract meaningful data from their extreme and distinct heterogeneities for use in the real-time diagnostics. The glycans on the surface of EVs could mark their subpopulations and establish the glycosignature, the solubilisation signature and molecular patterns. They all contribute to a new way of looking at and looking for composite biomarkers.

Antiphospholipid syndrome (APS) is a rare systemic autoimmune disease characterized by recurrent pregnancy morbidity or thrombosis in combination with the persistent presence of antiphospholipid antibodies (aPLs) in plasma/serum. Antiphospholipid antibodies are a heterogeneous, overlapping group of autoantibodies, of which anti-β2-glycoprotein I (aβ2GPI), anticardiolipin (aCL) antibodies and antibodies that prolong plasma clotting time in tests in vitro known as lupus anticoagulant (LAC) are included in the laboratory criteria for the diagnosis of APS. The presence of LAC antibodies in plasma is indirectly determined by measuring the length of coagulation in two tests - activated partial thromboplastin time (aPTT) and diluted Russell's viper venom time (dRVVT). The concentration of aβ2GPI and aCL (immunglobulin G (IgG) and immunoglobulin M (IgM) isotypes) in serum is directly determined by solid-phase immunoassays, either by enzyme-linked immunosorbent assay (ELISA), fluoroimmunoassay (FIA), immunochemiluminescence (CLIA) or multiplex flow immunoassay (MFIA). For patient safety, it is extremely important to control all three phases of laboratory testing, i.e. preanalytical, analytical and postanalytical phase. Specialists in laboratory medicine must be aware of interferences in all three phases of laboratory testing, in order to minimize these interferences. The aim of this review was to show the current pathophysiological aspects of APS, the importance of determining aPLs-a in plasma/serum, with an emphasis on possible interferences that should be taken into account when interpreting laboratory findings.

Microribonucleic acids (miRNAs) have emerged as a new category of biomarkers for many human diseases like cancer, cardiovascular and neurodegenerative disorders. MicroRNAs can be detected in various body fluids including blood, urine and cerebrospinal fluid. However, the literature contains conflicting results for circulating miRNAs, which is the main barrier to using miRNAs as non-invasive biomarkers. This variability in results is largely due to differences between studies in sample processing methodology, miRNA quantification and result normalization. The purpose of this review is to describe the various preanalytical, analytical and postanalytical factors that can impact miRNA detection accuracy and to propose recommendations for the standardization of circulating miRNAs measurement.

Introduction: Due to high inter-observer variability the 2015 International Council for Standardization in Haematology (ICSH) recommendations state to count band neutrophils as segmented neutrophils in the white blood cell (WBC) differential. However, the inclusion of bands as a separate cell entity within the WBC differential is still widely used in hematology laboratories in Croatia. The aim of this multicentric study was to assess the degree of inter-observer variability in enumerating band neutrophils within the WBC differential among Croatian laboratories.

Materials and methods: Seven large Croatian hospital laboratories from different parts of the country participated in the study. In each of 7 participating laboratories, one blood smear, that was flagged by the analyzer as possibly having bands, was evaluated by all personnel participating in the analysis of hematology samples. Between-observer manual smear reproducibility was expressed as coefficient of variation (CV) and calculated using the following formula: CV (%) = (standard deviation (SD)/mean value) x 100%.

Results: The CVs (%) and relative band neutrophil counts in participating laboratories were as follows: 15.4% (16-24), 19.2% (16-32), 19.5% (17-40), 21.1% (17-44), 35.0% (8-26), 51.9% (3-29), and remarkably high 62.4% (12-59). For segmented neutrophils CVs were lower, ranging from 7.4% to 32.2%. The CVs did not correlate with the number of staff members in each hospital (P = 0.293).

Conclusions: This study revealed very high variability in enumerating band neutrophil count in the blood smear differential among all participants, thus prompting a need for action on a national level.

Introduction: Glycomics, focusing on the role of glycans in biological processes, particularly their influence on the folding, stability and receptor interactions of glycoconjugates like antibodies, is vital for our understanding of biology. Changes in immunoglobulin G (IgG) N-glycosylation have been associated with various physiological and pathophysiological conditions. Nevertheless, time-consuming manual sample preparation is one of the limitations in the glycomics diagnostic implementation. The study aimed to develop an automated method for sample preparation on the Tecan Freedom Evo 200 platform and compare its efficiency and precision with the manual counterpart.

Materials and methods: The initial method development included 32 pooled blood plasma technical replicates. An additional 24 pooled samples were used in the method comparison along with 78 random duplicates of plasma samples collected from 10,001 Dalmatians biobank to compare the manual and automated methods.

Results: The development resulted in a new automated method. For the automated method, glycan peaks comprising 91% of the total sample glycan showed a variation of less than 5% while 92% of the total sample showed a variation of less than 5% for the manual method. The results of the Passing-Bablok regression indicated no differences between the automated and manual methods for 12 glycan peaks (GPs). However, for 8 GPs systematic difference was present, while both systematic and proportional differences were present for four GPs.

Conclusions: The developed automated sample preparation method for IgG glycan analysis reduced exposure to hazardous chemicals and offered a simplified workflow. Despite slight differences between the methods, the new automated method showed high precision and proved to be highly comparable to its manual counterpart.

This case report describes a case of pseudonormokalemia, true hypokalemia. Often, only laboratory values outside the normal range gain attention and false normal results are at risk of not being noticed. However, a disease state may be masked by another pathological process. Here, a 50-year old male was admitted to the Department of Internal Medicine due to sepsis from a dental infection. Initially, serum potassium measurement revealed a normal value of 4 mmol/L (reference interval 3.8-5.1 mmol/L). Thrombocyte number was above 500x10⁹/L. Due to our policy to recommend a repeated measurement of potassium in whole blood or heparin plasma if a patient has thrombocytosis, pseudonormokalemia was identified because the heparin plasma potassium value was only 2.9 mmol/L (reference interval 3.5-4.8 mmol/L). The physiological difference between serum and plasma concentration is no more than 0.3 mmol/L. In this case, potassium concentration were falsely elevated in the serum sample, probably caused by the high number of platelets releasing potassium during clotting. Interpretative comments in patients with thrombocytosis over 500x10⁹/L recommending plasma potassium measurement are helpful. The best way to eliminate pseudohyperkalemia and pseudonormokalemia phenomena caused by thrombocytosis is to completely change towards heparin plasma as the standard material.

Introduction: We compared the quality control efficiency of artificial intelligence-patient-based real-time quality control (AI-PBRTQC) and traditional PBRTQC in laboratories to create favorable conditions for the broader application of PBRTQC in clinical laboratories.

Materials and methods: In the present study, the data of patients with total thyroxine (TT4), anti-Müllerian hormone (AMH), alanine aminotransferase (ALT), total cholesterol (TC), urea, and albumin (ALB) over five months were categorized into two groups: AI-PBRTQC group and traditional PBRTQC group. The Box-Cox transformation method estimated truncation ranges in the conventional PBRTQC group. In contrast, in the AI-PBRTQC group, the PBRTQC software platform intelligently selected the truncation ranges. We developed various validation models by incorporating different weighting factors, denoted as λ. Error detection, false positive rate, false negative rate, average number of the patient sample until error detection, and area under the curve were employed to evaluate the optimal PBRTQC model in this study. This study provides evidence of the effectiveness of AI-PBRTQC in identifying quality risks by analyzing quality risk cases.

Results: The optimal parameter setting scheme for PBRTQC is TT4 (78-186), λ = 0.03; AMH (0.02-2.96), λ = 0.02; ALT (10-25), λ = 0.02; TC (2.84-5.87), λ = 0.02; urea (3.5-6.6), λ = 0.02; ALB (43-52), λ = 0.05.

Conclusions: The AI-PBRTQC group was more efficient in identifying quality risks than the conventional PBRTQC. AI-PBRTQC can also effectively identify quality risks in a small number of samples. AI-PBRTQC can be used to determine quality risks in both biochemistry and immunology analytes. AI-PBRTQC identifies quality risks such as reagent calibration, onboard time, and brand changes.

Introduction: Many studies report vitamin D (25-OH-D) deficiency, although there is no consensus among scientific societies on cut-offs and reference intervals (RI). The aim of this study is to establish and compare RI for serum 25-OH-D by direct and indirect methods.

Materials and methods: Two studies were performed in Zaragoza (Spain). A retrospective study (N = 7222) between January 2017 and April 2019 was used for RI calculation by indirect method and a prospective study (N = 312) with healthy volunteers recruited in August 2019 and February 2020 for direct method. Seasonal differences were investigated. Measurements were performed on Cobas C8000 (Roche-Diagnostics, Basel, Switzerland) using electrochemiluminescence immunoassay technology.

Results: Reference intervals (2.5-97.5 percentile and corresponding 95% confidence intervals, CIs) were as follows: by indirect method 5.6 ng/mL (5.4 to 5.8) - 57.2 ng/mL (55.2 to 59.8), in winter 5.4 ng/mL (5.2 to 5.7) - 55.7 ng/mL (53.6 to 58.4), while in summer 5.9 ng/mL (5.4 to 6.2) - 59.9 ng/mL (56.3 to 62.9). By direct method 9.0 ng/mL (5.7 to 9.5) - 41.4 ng/mL (37.6 to 48.0), in winter 7.4 ng/mL (3.9 to 8.6) - 34.6 ng/mL (30.6 to 51.5), while in summer 13.3 ng/mL (10.1 to 14.1) - 44.1 ng/mL (38.9 to 66.0). In both methods, RIs were higher in summer. A significant difference was observed in 25-OH-D median values between the two methods (P < 0.001).

Conclusions: Reference interval calculation according to the studied area may be a useful tool to adapt the deficiency cut-offs for 25-OH-D. Our data support 25-OH-D values over 12.0 ng/mL for healthy population as sufficient, therefore current recommendations should be updated. In addition, differences in seasonality should be taken into account.