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Although the use of cryoprotectant dimethyl sulfoxide (DMSO) is the gold standard in cryopreservation of hematopoietic stem cells, it is well known that it has a negative effect on cell viability. The aim of this prospective study was to examine how the length of post-thaw exposure to DMSO affects the cell viability and stability of peripheral blood stem cell (PBSC) samples. Additionally, the effects of donor type and pre-cryopreservation storage time on post-thaw viability during the stability study were evaluated. In 30 autologous and 30 allogeneic PBSC samples viable CD34+, CD14+, CD19+, CD16+/56+, and CD3+ cells were determined immediately after thawing, and one-and three-hours post-thaw.

Background/Objectives: This study assessed the effects of 5G radiofrequency electromagnetic radiation (RF-EMR) at different frequencies (700 MHz, 2500 MHz, 3500 MHz) on the complete blood count (CBC), erythrocyte morphometry, and platelet activation after the short-term in vitro exposure of human blood. Methods: Blood samples from 30 healthy volunteers (15 men and 15 women, aged 25–40 years old) were collected at three intervals (14 days apart). For each collection, four tubes of blood were drawn per volunteer—two experimental and two controls. Experimental samples were exposed to 5G RF-EMR for 2 h at room temperature using a half-cone gigahertz transverse electromagnetic cell. The CBC was analysed via a haematology analyser, the erythrocyte morphometry was analysed using the SFORM program, and platelet activation was analysed via flow cytometry. Results: The CBC and platelet activation showed no significant differences between the experimental and control samples. However, the erythrocyte morphometry exhibited notable changes. At 700 MHz, the erythrocyte size, contour, and membrane roughness increased significantly for both sexes, with women's cells showing greater sensitivity. At 2500 MHz, women exhibited an increased contour index and a decreased solidity and form factor. At 3500 MHz, women showed an increased contour index and outline but a decreased solidity, elongation, and form factor. Cluster analysis identified two erythrocyte subpopulations: smaller, rounder cells with smooth membranes and larger cells with rougher membranes. Conclusions: These results indicate that 5G RF-EMR exposure significantly alters erythrocyte morphometry. The strongest effects were observed at 700 MHz, where men exhibited greater membrane roughness, and women showed larger and rounder erythrocytes. These findings suggest that short-term in vitro 5G RF-EMR exposure disrupts the cytoskeleton, increasing membrane permeability and deformability. [ABSTRACT FROM AUTHOR]

Autovalidation is a computerised postanalytical tool that uses a sequence of procedures to verify laboratory test results without manual intervention. The Working Group for Post-analytics of the Croatian Society for Medical Biochemistry and Laboratory Medicine has prepared procedures for the implementation of autovalidation in routine laboratory work, which complement the existing national recommendations and aim to clarify the procedures of autovalidation. Before implementation, it is necessary to determine the need for the introduction of autovalidation in routine laboratory work, and then appoint the autovalidation team, whose task is to decide in which area of laboratory work autovalidation should be introduced, create the algorithm and supervise the verification of autovalidation. Standard rules included in the algorithm are patient data, messages from the analyzer, values of interference indices, autovalidation range and delta check. All criteria defined in the autovalidation algorithm have to be documented and approved by the laboratory manager. This autovalidation procedure shows the basic rules of autovalidation that can be used by any laboratory in the initial phase. The justification for using autovalidation will depend on the number and complexity of laboratory tests, the size of the laboratory personnel, and the available financial and material resources. Autovalidation avoids the subjective evaluation of laboratory test results as it is based on the same rules and is standardised to a certain extent, which further increases the quality of laboratory test results. [ABSTRACT FROM AUTHOR]

ABSTRACT Introduction Methods Results Conclusions The standard flow cytometry method for viability testing using 7‐aminoactinomycin D (7‐AAD) determines cells in necrosis and late apoptosis. The colony‐forming unit (CFU) assay, which evaluates the proliferation ability of HSCs, is also used in graft quality assessment despite known deficiencies that make this assay impractical in routine clinical settings. The aim was to compare the effectiveness of the flow cytometry 7‐AAD/annexin V method with the 7‐AAD method in assessing the quality of HSCs in autologous and allogeneic peripheral blood stem cell (PBSC) products.Thirty autologous and 30 allogeneic fresh and thawed cryopreserved PBSC products were included in this study. The viability of HSCs was determined using the 7‐AAD method and 7‐AAD/annexin V method on a flow cytometer, while their clonogenic capacity was assessed by CFU assay.There was an excellent correlation for CD34+ cell viability between the 7‐AAD and the 7‐AAD/annexin V method for fresh samples (Rs = 0.930, p < 0.001) and a good correlation for thawed PBSC samples (Rs = 0.739, p < 0.001). Excellent correlation was observed for post‐thaw CD34+ cell recovery between the two methods for viability (Rs = 0.980, p < 0.001). Statistical analysis showed a weak correlation between CFU‐GM recovery and CD34+ cell recovery, regardless of which viability testing method was used (7‐AAD method p = 0.021, Rs = 0.298; 7‐AAD/annexin V method p = 0.029, Rs = 0.282).Results of this study showed that in the quality assessment of cryopreserved PBSC product viability, the 7‐AAD/annexin V method had no added value compared to the 7‐AAD method, which was suitable enough for routine quality control of cryopreserved autologous and allogeneic PBSC samples. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor-T (CAR-T) cell therapy is currently the best-known type of immune effector cells therapy. For CAR T-cell therapy, the determination of CD3+ T cells is necessary for the quality control of fresh leukapheresis product as starting material. The aim was to validate analytical method for quantification of percentage and absolute count of T lymphocyte subtypes (CD3+, CD4+ and CD8+ cells) in fresh apheresis products using single-platform method on flow cytometer BD FACS Canto II. Validation study included determination of precision, trueness (bias), assessment of linearity, carryover, comparison of results obtained with two different protocols on flow cytometer for CD3+ cells determination and stability study. For between-run precision coefficients of variation (CVs) were <20%, as well as bias for all T-lymphocyte subtypes. For within-run precision, CVs were <10%, except for low CD8+ cell (percentage 10.51% and viable absolute count 12.37%). Comparison of results obtained with two different protocols for CD3+ cells determination shows no statistically significant difference. Statistically significant differences between results of the analysis of CD4+ cells in fresh samples and results obtained after storage at 4 °C (p =.004) and at room temperature (p =.018) were found. In conclusion, method for enumeration of T-lymphocyte subtypes can be used in routine work on BD FACS Canto II instrument for quality assessment of fresh cell products collected by leukapheresis procedure. [ABSTRACT FROM AUTHOR]

Cellular therapy nowadays includes various products from haematopoietic stem cells (HSC) collected from bone marrow, peripheral blood, and umbilical cord blood to more complex adoptive immune therapy for the treatment of malignant diseases, and gene therapy for inherited immune deficiencies. Broader utilization of cellular therapy requires extensive quality testing of these products that should fulfil the same requirements regarding composition, purity, and potency nevertheless they are manufactured in various centres. Technical improvements of the flow cytometers accompanied by the increased number of available reagents and fluorochromes used to conjugate monoclonal antibodies, enable detailed and precise insight into the function of the immune system and other areas of cell biology, and allows cell evaluation based on size, shape, and morphology or assessment of cell surface markers, as well as cell purity and viability, which greatly contributes to the development and progress of the cell therapy. The aim of this paper is to give an overview of the current use and challenges of flow cytometry analysis in quality assessment of cellular therapy products, with regard to basic principles of determining HSC and leukocyte subpopulation, assessment of cells viability and quality of thawed cryopreserved HSC as well as the importance of validation and quality control of flow cytometry methods according to good laboratory practice. [ABSTRACT FROM AUTHOR]

Introduction: Interpretation of laboratory test results is a complex post-analytical activity that requires not only understanding of the clinical significance of laboratory results but also the analytical phase of laboratory work. The aims of this study were to determine: 1) the general opinion of Croatian medical biochemistry laboratories (MBLs) about the importance of interpretative comments on laboratory test reports, and 2) to find out whether harmonization of interpretative comments is needed. Materials and methods: This retrospective study was designed as a survey by the Working Group for Post-analytics as part of national External Quality Assessment (EQA) program. All 195 MBLs participating in the national EQA scheme, were invited to participate in the survey. Results are reported as percentages of the total number of survey participants. Results: Out of 195 MBLs, 162 participated in the survey (83%). Among them 59% MBLs implemented test result comments in routine according to national recommendations. The majority of laboratories (92%) state that interpretative comments added value to the laboratory reports, and a substantial part (72%) does not have feedback from physicians on their significance. Although physicians and patients ask for expert opinion, participants stated that the lack of interest of physicians (64%) as well as the inability to access patient's medical record (62%) affects the quality of expert opinion. Conclusion: Although most participants state that they use interpretative comments and provide expert opinions regarding test results, results of the present study indicate that harmonization for interpretative comments is needed. [ABSTRACT FROM AUTHOR]

Introduction: The detection and prevention of errors in the postanalytical phase can be done through the harmonization and standardization of constituent parts of this phase of laboratory work. The aim was to investigate how well the ongoing management of the postanalytical phase corresponds to the document "Post-analytical laboratory work: national recommendations" in Croatian medical biochemistry laboratories (MBLs). Materials and methods: All 195 MBLs participating in the national external quality assessment scheme, were invited to undertake a part in a survey. Through 23 questions the participants were asked about management of the reference intervals (RI), delta check, reflex/reflective testing, postanalytical quality indicators and other parts of the postanalytical phase recommended in the national recommendations. The results are presented in numbers and percentages. Results: Out of 195 MBLs, 119 participated in the survey, giving a response rate of 61%. Not all of the respondents provided answers to all the questions. Delta check has not been used in 59% (70/118) of the laboratories. Only 22/113 (20%) laboratories use reflex and/or reflective testing. In 53% of the laboratories, critical results were reported within 30 minutes of the confirmation of the results. In 34% (40/118) of the laboratories, turnaround time and reporting of critical results are two most often monitored postanalytical quality indicators. Conclusion: The results showed the critical results reporting and monitoring of postanalytical quality indicators are in the line with the recommendations. However, the management of RI verification, the use of delta check and reflex/reflective testing still must be harmonized among Croatian MBLs. [ABSTRACT FROM AUTHOR]

Introduction: Autovalidation (AV) is an algorithm based on predefined rules designed, among others, to automate and standardize the postanalytical phase of laboratory work. The aim of this study was to examine the overall opinion of Croatian medical biochemistry laboratories regarding various aspects of AV. Material and methods: This retrospective study is an analysis of the responses of a survey about AV comprised of 18 questions, as part of Module 10 ("Postanalytical phase of laboratory testing") of national External Quality Assessment program, administered by the Croatian Centre for Quality Assessment in Laboratory Medicine. Results were reported as percentages of total number of participants in survey or as proportions of observed data if the overall number of data was <100. Results: 121 laboratories responded to the survey, of which 76% do not use AV, while 11% of laboratories use AV in routine laboratory work. 16/29 laboratories implemented semi-automated AV for general biochemistry (7/29), haematology (5/29), and coagulation (4/29) tests. Analytical measurement ranges, critical values, flags from analysers, interference indices and delta check were the most commonly used rules in the algorithm. 12/29 laboratories performed validation of AV with less than 500 samples (8/29). 7/13 laboratories report the percentage of AV being 20-50%, while 10/13 answered that introduction of AV significantly reduced turnaround time (TAT) (for 20 - 25%), especially for biochemistry tests. Conclusions: Despite of its numerous benefits (i.e. shorter TAT, less manual validation, standardization of the postanalytical phase), only a small number of Croatian laboratories use AV. [ABSTRACT FROM AUTHOR]

Introduction: CD34+ hematopoietic stem cell (HSC) enumeration by cell flow cytometry is routinely used in clinical laboratories for monitoring of HSC mobilization into peripheral blood and assessment of the quality of HSC products. The modified ISHAGE protocol is the most often used procedure for determination of CD34+ cells using flow cytometry. The aim of this study was to evaluate BD Enumeration stem cell kit on flow cytometer BD facscanto II, using facscanto clinical and facsdiva softwares. Methods: Validation study included determination of within‐run and between‐run precision, trueness (bias), comparison of the test results analyzed on facscanto clinical and facsdiva softwares, assessment of linearity, specimen stability, and carryover. Results: For between‐run precision, coefficients of variation (CVs) were all <10%, except for low control level on facsdiva software. CVs for within‐run precision were <10%, except for high absolute count of CD34+ cells on facsdiva software. Comparison of data showed no statistically significant differences between facscanto clinical and facsdiva software (Spearman's rank correlation coefficients were.993 for % of CD34+ cells and 0.983 for absolute count of CD34+ cells). In linearity study, bias for all dilutions was < 20%, and carryover assessment cannot be considered significant on both softwares. There was a statistically significant difference (P =.044) in absolute count of CD34+ cells after 24 hours of storage, when using facscanto clinical software. Conclusion: BD Stem Cell Enumeration Kit can be used in routine laboratory work on BD FACSCanto II instrument, whereas facscanto clinical and facsdiva software were used for acquisition and data analysis. [ABSTRACT FROM AUTHOR]

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Objective Autovalidation algorithm should be properly designed with clearly defined criteria and any data that do not meet the criteria, must be reviewed and manually validated. The aim was to define the rules for autovalidation in our laboratory information system (LIS), and validate the algorithm prior to its implementation in routine laboratory work. Methods Autovalidation was implemented for all routine serum biochemistry tests. The algorithm included analytical measurement ranges (AMR), delta check, critical values, serum indices and all preanalytical and analytical flags from the analyzer. Results In the validation process 9805 samples were included, and 78.3% (7677) of all samples were autovalidated. The highest percentage of non-validated samples (54.9%) refers to those with at least one result outside the method linearity ranges (AMR criteria) while critical values were observed to be the least frequent criterion for stopping autovalidation (1.8%). Also, 38 samples were manually validated as they failed to meet the autovalidation criteria. Conclusion Implementation of algorithm for autovalidation in our institution resulted in the redesign of the existing LIS. This model of the autovalidation algorithm significantly decreased the number of manually validated test results and can be used as a model for introducing autovalidation in other laboratory settings. [ABSTRACT FROM AUTHOR]

Bacterial infections and sepsis are major problems in critically ill patients. Timely diagnosis and therapy reduce morbidity and mortality. Many studies have included the investigation of various biomarkers whose elevated concentrations can indicate sepsis; among them, PCT proved to be most useful. PCT is synthesized in the thyroid gland as a prohormone of calcitonin. In healthy individuals the PCT concentration is <0.1 ng/mL. The advantage of the PCT is a high negative predictive value for the exclusion of sepsis, with the cut-off value of 0.5 ng/ml. A concentration between 2 and 10 ng/ml indicates strong sepsis, whereas a value .10 ng/ml is associated with septic shock. In addition to the diagnosis of sepsis, the measurement of PCT concentration is useful for the introduction and monitoring of antibiotic therapy, which is performed according to an algorithm based on the cutoff value for PCT. Immunoassays are used to measure PCT concentrations in serum or plasma. It is possible to determine the concentration in whole blood by using point-of-care testing. In pathological conditions that are not associated with sepsis, PCT is useful as a prognostic indicator of disease complications. Some studies suggest that PCT is a potential early indicator of acute coronary syndrome. [ABSTRACT FROM AUTHOR]

The post-analytical phase is the final phase of the total testing process and involves evaluation of laboratory test results; release of test results in a timely manner to appropriate individuals, particularly critical results; and modification, annotation or revocation of results as necessary to support clinical decision-making. Here we present a series of recommendations for post-analytical best practices, tailored to medical biochemistry laboratories in Croatia, which are intended to ensure alignment with national and international norms and guidelines. Implementation of the national recommendations is illustrated through several examples. [ABSTRACT FROM AUTHOR]