Optimal Sample Preparation Conditions Research Articles

Determination of selenium in polyheteroatomic organic compounds by microwave plasma atomic emission spectrometry

Currently, the problem of selenium determination in various objects attracts a large number of scientists. Interest in this area is attributed to the biological importance of selenium, since it is both an important nutrient and has a toxic effect on the body, depending on its amount and the properties of the compounds in which it is contained. Checking the purity of selenium-containing organic compounds (both determination of impurities and analysis of the basic composition), which is carried out by methods of elemental analysis, is an important task, however, in modern literature there is lack of information regarding this problem. The purpose of the presented study is to develop a method for the determination of selenium in the basic composition of synthetic polyheteroatomic organic compounds by microwave plasma atomic emission spectrometry. Various selenodiazole derivatives were mainly used for analysis. Optimal conditions for sample preparation and atomic emission determination of the selenium content were specified. To mineralize the substances under study, oxygen flask combustion was used with the addition of potassium nitrate, which improves combustion and prevents the formation of soot. Hydrochloric acid at a concentration of 0.1 M was used as an absorption solution. It is noted that before measurements it is necessary to purge the monochromator and optimize the pressure in the atomic emission spectrometer nebulizer and optimize pressure in the atomizer. In addition, the number of replicates and sampling time were increased to optimize selenium measurement. Under specified conditions, the metrological characteristics of the technique were determined: the detection limit and quantitation limit, intra-laboratory precision and accuracy. Selenium was determined in 11 synthetic organic compounds with selenium content ranged from 12 to 51%. The error of the analysis did not exceed 0.3%, which meets the requirements of organic elemental analysis.

Matrix-assisted laser desorption/ionization matrix incorporation evaluation algorithm for improved peak coverage and signal-to-noise ratio in mass spectrometry imaging.

Despite decades of implementation, the selection of optimal sample preparation conditions for matrix-assisted laser desorption/ionization (MALDI) imaging is still ambiguous due to the lack of a universal and comprehensive evaluation methodology. Thus, numerous experiments with different matrix application conditions accompany a translation of the method to novel sample types and matrices. Mouse brain tissues were covered with 9-aminoacridine through sublimation, followed by recrystallization in vapors of 5% (v/v) methanol solution in water. The samples were analyzed by MALDI time-of-flight mass spectrometry, and the efficiency of lipid and small-molecule ionization was evaluated with different metrics. We first investigate the dependency of matrix density and recrystallization conditions on the thickness of an analyte-empty matrix layer to roughly evaluate the laser shot number required to obtain an intense signal with minimal noise. Then, we introduce metrics for the analysis of small imaging datasets (small sample regions) of model samples based on median quantity of peaks in spectra (medQP) and weighted median signal-to-noise ratio (wmSNR). The evaluation of small regions and taking median values for metrics help overcome the sample heterogeneity and allow for the simultaneous comparison of different acquisition parameters. Here, we propose a methodology based on gradual laser ablation of small regions of sample and further implementation of weighted signal-to-noise ratio to assess various matrix application conditions. The proposed approach helps reduce the number of test samples required to determine optimal sample preparation conditions and improve the overall quality of images.

SELECTION OF METHODOLOGY FOR THIOPHAN M ISOLATION FROM ANIMAL ORGANS

Introduction. An important preparatory stage of collecting information for the registration of a new drug is the study of its pharmacokinetic parame-ters in accordance with the requirements of good laboratory practice. The preparatory stage of such a study consists in the selection of optimal condi-tions for sample preparation of biological objects to determine the test substance in them. The purpose of this study is to select optimal conditions for sample preparation, including the pH value of the extractant, and isolation of Thio-phane M from the organs of laboratory animals (rats) for its further voltammetric determination according to the previously developed method. Material and methods. The object of the study was the substance Thiophane M [dodecyl(3,5,-dimethyl-4-hydroxybenzyl)sulfide], which is a promis-ing biologically active compound with antioxidant properties, developed on the basis of the Department of Chemistry of Novosibirsk State Pedagogical University together with the Research Institute of Antioxidant Chemistry. Experimental data were obtained using 5 male rats weighing from 350-380 g, which were injected with an intragastrically tested sample of Thiophane M at a dose of 500 mg/kg in the form of an oil solution in olive oil. Results. The total number of samples obtained and examined is 180. Based on the data obtained with their subsequent statistical processing by the indirect differences method according to the State Pharmacopoeia of the XIV edition, it was established that the optimal condition for the extraction of Thiophane M is a single extraction with diethyl ether, which is confirmed by the results of voltammetric analysis. We have found that the greatest number Thiophane M localized in the brain, heart and lungs (364.08.20, 332.016.47 and 275.025.40 µcg/g, respectively), fewer Thiophane M detect-ed in kidney, spleen and liver (146.015.50, 81.08.66 56.014.53 µcg/g, respectively). Conclusions. Of all the methods of Thiophan M isolation from rat organs, the method using diethyl ether as an extractant was chosen, which gave stable results due to the maximum extraction of the substance.

Multiparameter flow cytometric detection and analysis of rare cells in in vivo models of cancer metastasis.

Rapid and reliable circulating tumor cell (CTC) and disseminated tumor cell (DTC) detection are critical for rigorous evaluation of in vivo metastasis models. Clinical data show that each step of the metastatic cascade presents increasing barriers to success, limiting the number of successful metastatic cells to fewer than 1 in 1,500,000,000. As such, it is critical for scientists to employ approaches that allow for the evaluation of metastatic competency at each step of the cascade. Here, we present a flow cytometry-based method that enables swift and simultaneous comparison of both CTCs and DTCs from single animals, enabling evaluation of multiple metastatic steps within a single model system. We present the necessary gating strategy and optimized sample preparation conditions necessary to capture CTCs and DTCs using this approach. We also provide proof-of-concept experiments emphasizing the appropriate limits of detection of these conditions. Most importantly, we successfully recover CTCs and DTCs from murine blood and bone marrow. In Supplemental materials, we expand the applicability of our method to lung tissue and exemplify a potential multi-plexing strategy to further characterize recovered CTCs and DTCs. This approach to multiparameter flow cytometric detection and analysis of rare cells in in vivo models of metastasis is reproducible, high throughput, broadly applicable, and highly adaptable to a wide range of scientific inquiries. Most notably, it simplifies the recovery and analysis of CTCs and DTCs from the same animal, allowing for a rapid first look at the comparative metastatic competency of various model systems throughout multiple steps of the metastatic cascade.

Greening of the method for simultaneous determining the enisamium iodide and tilorone dihydrochloride using GC-FID assay

Pharmaceutical companies in Ukraine aspire to develop their innovative medicinal products and successfully introduce them to the global market. However, along with the prospects of increased usage of these pharmaceuticals, there arises a challenge of heightened waste production, making them a part of the over twenty million tons of PPCPs produced annually. Consequently, one of the tasks in producing new pharmaceuticals is the development of methodologies and approaches not only for quality control but also for their determination in the environment matrices.
 The aim. Develop and validate GC-FID chromatographic method for the simultaneous determination of Enisamium iodide and Tilorone dihydrochloride, evaluate their applicability, and compare their "greenness" with the previously developed HPLC method.
 Materials and methods. The determination of the Tilorone dihydrochloride and Enisamium iodide was carried out by gas chromatography with a flame ionization detector using the Rxi-5 ms (30 m long, 0.25 mm outer diameter and 0.25 μm liquid stationary phase thickness)
 Results. Chromatographic GC-FID methods have been developed for the simultaneous determination of Enisamium iodide and Tilorone dihydrochloride. Optimal sample preparation conditions were established, and a validation process was conducted. A comparison with the previously developed HPLC method was made regarding "greenness."
 Conclusions. The developed GC-FID methodology is accurate and more environmentally friendly compared to the previously established methods. It can be recommended to determine Enisamium iodide and Tilorone dihydrochloride in the environmental matrices. It is considered environmentally friendly based on the overall GREENness (AGREE) scale, scoring 0.73 (>0.70), which demonstrates the environmentally favourable nature of the proposed analytical approach

Study of sample preparation influence on bacterial lipids profile in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Lipids are one of the cell components therefore it is important to be able to accurately assess them. One of the analytical techniques used to study lipid profiles is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The present study attempted to select optimal conditions for sample preparation and MALDI MS analysis of bacterial lipidome in both positive and negative ion modes using different extraction protocols-Folch, Matyash, and Bligh & Dyer, solvents used to apply samples, and matrices such as 9-aminoacridine (9-AA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), 2-mercaptobenzothiazole (MBT), and 2,4,6-trihydroxyacetophenone (THAP). The obtained results allowed concluding that DHB or CHCA matrices are suitable for lipid analysis in the positive mode, and in the negative mode THAP or 9-AA. The most appropriate protocol for extracting lipids from bacterial cells was the Bligh & Dyer method in both ionization modes. The use of the solvent TA30, which was a mixture of acetonitrile and 0.1% trifluoroacetic acid in water, provided on the spectra a significant number of signals from lipids in all groups analyzed, such as fatty acyls, glycerolipids, and glycerophospholipids.

Feasibility of Optical Genome Mapping from Placental and Umbilical Cord Sampled after Spontaneous or Therapeutic Pregnancy Termination

Optical genome mapping (OGM) is an alternative to classical cytogenetic techniques to improve the detection rate of clinically significant genomic abnormalities. The isolation of high-molecular-weight (HMW) DNA is critical for a successful OGM analysis. HMW DNA quality depends on tissue type, sample size, and storage conditions. We assessed the feasibility of OGM analysis of DNA from nine umbilical cord (UC) and six chorionic villus (CV) samples collected after the spontaneous or therapeutic termination of pregnancy. We analyzed quality control metrics provided by the Saphyr system (Bionano Genomics) and assessed the length of extracted DNA molecules using pulsed-field capillary electrophoresis. OMG data were successfully analyzed for all six CV samples. Five of the UC samples did not meet the Saphyr quality criteria, mainly due to poor DNA quality. In this regard, we found that DNA quality assessment with pulsed-field capillary electrophoresis can predict a successful OGM analysis. OGM data were fully concordant with the results of standard cytogenetic methods. Moreover, OGM detected an average of 14 additional structural variants involving OMIM genes per sample. On the basis of our results, we established the optimal conditions for sample storage and preparation required for a successful OGM analysis. We recommend checking DNA quality before analysis with pulsed-field capillary electrophoresis if the storage conditions were not ideal or if the quality of the sample is poor. OGM can therefore be performed on fetal tissue harvested after the termination of pregnancy, which opens up the perspective for improved diagnostic yield.

Suspect Screening of Lupin-Produced Phytotoxins in Environmental Samples

Phytotoxins, secondary metabolites synthesized by plants, are now recognized as a new category of environmental micropollutants. Only a limited number of phytotoxins have been detected and reported in terrestrial and aquatic environments, partially due to analytical challenges. In this article, a robust, reliable, and efficient approach, namely Source Supported Suspect Screening (4S), is introduced for high-throughput analysis of phytotoxins from their plant origin to the downstream environmental compartments. The approach was established on an ultrahigh-performance liquid chromatography–electrospray ionization–quadrupole‑time‑of‑flight-mass spectrometry (UHPLC–ESI–QTOF-MS) analytical platform where optimal sample preparation and chromatographic conditions were investigated and a new suspect screening protocol was suggested.

Evaluating the use of locked nucleic acid capture probes in hybrid LC-MS/MS analysis of siRNA analytes.

Background: Hybrid LC-MS assays for oligonucleotides rely on capture probes to develop assays with high sensitivity and specificity. Locked nucleic acid (LNA) probes are thermodynamically superior to existing capture probes, but are not currently used for hybrid LC-MS assays. Materials & methods: Using two lipid-conjugated double-stranded siRNAcompounds as model analytes, hybrid LC-MS/MS assays using LNA probes were developed. Results: The workflows demonstrated the superiority of the LNA probes, optimized sample preparation conditions to maximize analyte recovery, evaluated the need for analyte-specific internal standards, and demonstrated that advanced mass spectrometric technology can increase assay sensitivity by up to 20-fold. Conclusion: The workflow can be used in future bioanalytical studies to develop effective hybrid LC-MS/MS methods for siRNA analytes.

Preparation of a Single-Cell Suspension from Tumor Biopsy Samples for Single-Cell RNA Sequencing.

An essential requirement for single-cell RNA sequencing in cancer is the preparation of high-quality single-cell suspensions from the tumor tissue. In this work, various methods of dissociation of tumor biopsy specimens were analyzed and developed to obtain a cell suspension with at least 80% viability. It was found that the optimal conditions for sample preparation are mechanical dissociation followed by incubation with a collagenase/hyaluronidase mixture with addition of DNAase I for 60 min. Thus, we optimize the approach for preparing single-cell suspensions from the tumor biopsy tissue for single-cell RNA sequencing.

Development and validation of a peptide-mapping procedure for a novel C1 esterase inhibitor

Peptide mapping is a key method for studying the primary structure of proteins. With its sensitivity to the slightest changes in the covalent structure of a protein, this method is applicable both to medicinal product identification at the control stage and to production process stability monitoring.The aim of the study was to develop and validate a peptide-mapping procedure for the identification of a novel highly glycosylated recombinant C1 esterase inhibitor.Materials and methods. The authors studied recombinant human C1 esterase inhibitor products and trypsin. The study involved peptide mapping using reverse-phase high-performance liquid chromatography and high-resolution mass spectrometry. The following statistics were calculated to evaluate the results: mean, standard deviation, and coefficient of variation. The validation parameters included specificity, precision, and robustness.Results. The authors tested several variants of sample preparation for tryptic digests, including additional N-glycanase treatment and complete deglycosylation, and established the optimal conditions for sample preparation and chromatographic separation of C1 esterase inhibitor peptides to obtain consistent chromatographic profiles (peptide maps). The authors identified characteristic peaks and the corresponding relative retention time and area ranges. The absolute retention time of the second (characteristic) peak was approximately 16.5–16.9 minutes. The relative retention times were 2.14–2.21 for peak 9, 2.55–2.64 for peak 12, 2.97–3.14 for peak 14, 3.11–3.29 for peak 15, and 6.20–6.63 for peak 28.Conclusions. The authors developed a peptide-mapping procedure for C1 esterase inhibitors and optimised the conditions to achieve an over 18-hour reduction in sample preparation time. This procedure met the established acceptance criteria for specificity, precision, and robustness.

Suspect Screening of Lupin-Produced Phytotoxins in Environmental Samples

Phytotoxins, the secondary metabolites synthesized by plants, are now recognized as a new category of environmental micropollutants. So far, only a limited number of phytotoxins have been detected and reported in terrestrial and aquatic environments, partially due to the analytical challenge involved. In this article, a robust, reliable, and efficient approach, namely Source Supported Suspect Screening (4S), is introduced for high-throughput analysis of phytotoxins from their plant origin to the downstream environmental compartments. The approach was established on an ultrahigh-performance liquid chromatography–electrospray ionization–quadrupole‑time‑of‑flight-mass spectrometry (UHPLC–ESI-QTOF-MS) analytical platform where optimal sample preparation and chromatographic conditions were investigated and a new suspect screening protocol was suggested.

Development of Acid Hydrolysis-Based UPLC-MS/MS Method for Determination of Alternaria Toxins and Its Application in the Occurrence Assessment in Solanaceous Vegetables and Their Products.

In this work, we proposed an acid hydrolysis-based analytical method for the detection of Alternaria toxins (ATs) in solanaceous vegetables and their products with solid-phase extraction (SPE) and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This study was the first to reveal that some compounds in the eggplant matrix bind to altenusin (ALS). Validation under optimal sample preparation conditions showed that the method met the EU criteria, exhibiting good linearity (R2 > 0.99), matrix effects (-66.6--20.5%), satisfying recovery (72.0-107.4%), acceptable precision (1.5-15.5%), and satisfactory sensitivity (0.05-2 µg/kg for limit of detection, 2-5 µg/kg for limit of quantification). Out of 393 marketed samples, only 47 samples were detected, ranging from 0.54-806 μg/kg. Though the occurrence ratio (2.72%) in solanaceous vegetables could be negligible, the pollution status in solanaceous vegetable products was much more serious, and the incidences were 41.1%. In the 47 contaminated samples, the incidences were 4.26% for alternariol monomethyl ether (AME), 6.38% for alternariol (AOH) and altenuene (ALT), 42.6% for tentoxin (TEN), and 55.3% for tenuazonic acid (TeA).

Mathematical planning of the liquid-liquid extraction procedure: a forensic chemical study of verapamil in blood

The objective of the study is to determine the optimal conditions for blood sample preparation by liquid-liquid extraction using mathematical planning of the experiment by the example of forensic chemical study of verapamil. Evaluation of verapamil recovery from blood samples was performed according to the scheme of multivariate three-level experimental planning using the Box-Behnken design. The effect of aqueous phase pH, the ratio of chloroform to n-butanol in the extraction agent, extraction and centrifugation time, addition of sodium chloride, and the ratio of sample and extraction agent volumes on the recovery was studied. The algorithm using mathematical planning and development of liquid-liquid extraction model of target analyte can be recommended for analysis procedure optimization in forensic chemistry.

Simultaneous GC-NCI-MS Determination of Persistent Organic Pollutants and Current-Use Pesticides in Breast Milk Samples

To preserve human health, persistent organic pollutants (POPs) and current-use pesticides (CUPs) should be monitored in fatty tissues, including breast milk. Therefore, this study aimed to optimize sample preparation conditions using a 32 factorial design for the determination of POPs and CUPs by gas chromatography coupled to mass spectrometry with negative chemical ionization (GC-NCI-MS). The method was validated for 57 POP and CUP compounds using hexane:acetone for extraction and clean-up by dispersive solid phase extraction (d-SPE) with Florisil®. The matrix effect was compensated by extracted analytical calibration. Method validation showed satisfactory results with limits of detection of 3 to 13 ng g-1 of fat. The method presented adequate accuracy (recoveries from 72 to 117%) and precision (relative standard deviation (RSD) ≤ 18%) and was applied to breast milk samples from Rio Grande do Sul State, southern Brazil, wherein all samples contained at least one compound. With principal component analysis, it was possible to associate the pesticides detected with the city of origin of the samples and the number of pregnancies of nursing mothers. Additionally, the analytical method was effective for the determination of trace levels of POPs and CUPs in breast milk and can be applied in biomonitoring studies.

Determination of zinc by atomic absorption in automobile tire rubber as a source of ecotoxicants in atmospheric air

Annotation. A methodology for determining zinc in tires (as a source of ecotoxicants in the atmospheric air) by atomic absorption with microwave pre-treatment as a product of motor vehicle wear in St. Petersburg was developed. In the article, the author gives different ways of sampling for testing. One of the rubber samples was taken from one area no larger than 5x5 cm on one tyre (first sampling option), the second sample was taken cumulatively from all tyre sides around its circumference (second sampling option). Tests of selected samples were carried out allowing conclusions to be drawn about the optimal sampling method. A comparative analysis has been made of the test results of samples taken by different methods from the car tyre surface. Based on the comparative analysis conclusions were made about the homogeneity of the tire composition. Various physical and chemical methods of sample preparation for the heavy metals determination are presented. In the course of the work the author selected the optimal conditions for sample preparation for testing allowing to obtain the maximum possible extraction of the analyte at the stage of sample preparation. The atomic absorption method was used to determine the zinc content in automobile tire samples. On the basis of the results obtained conclusions about the optimal sampling methods and sample preparation were made and the test conditions of the samples were optimized.

Determination of zinc by atomic absorption in automobile tire rubber as a source of ecotoxicants in atmospheric air

Annotation. A methodology for determining zinc in tires (as a source of ecotoxicants in the atmospheric air) by atomic absorption with microwave pre-treatment as a product of motor vehicle wear in St. Petersburg was developed. In the article, the author gives different ways of sampling for testing. One of the rubber samples was taken from one area no larger than 5x5 cm on one tyre (first sampling option), the second sample was taken cumulatively from all tyre sides around its circumference (second sampling option). Tests of selected samples were carried out allowing conclusions to be drawn about the optimal sampling method. A comparative analysis has been made of the test results of samples taken by different methods from the car tyre surface. Based on the comparative analysis conclusions were made about the homogeneity of the tire composition. Various physical and chemical methods of sample preparation for the heavy metals determination are presented. In the course of the work the author selected the optimal conditions for sample preparation for testing allowing to obtain the maximum possible extraction of the analyte at the stage of sample preparation. The atomic absorption method was used to determine the zinc content in automobile tire samples. On the basis of the results obtained conclusions about the optimal sampling methods and sample preparation were made and the test conditions of the samples were optimized.

Standardized Workflow for Precise Mid- and High-Throughput Proteomics of Blood Biofluids.

Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that can accommodate 3 blood-based proteomes: naive plasma, depleted plasma and dried blood. Optimal conditions for sample preparation and data independent acquisition-mass spectrometry analysis were established in plasma then automated for depleted plasma and dried blood. The mass spectrometry workflow was modified to facilitate sensitive high-throughput analysis or deeper profiling with mid-throughput analysis. Analytical performance was evaluated by the linear response of peptides and proteins to a 6- or 7-point dilution curve and the reproducibility of the relative peptide and protein intensity for 5 digestion replicates per day on 3 different days for each biofluid. Using the high-throughput workflow, 74% (plasma), 93% (depleted), and 87% (dried blood) displayed an inter-day CV <30%. The mid-throughput workflow had 67% (plasma), 90% (depleted), and 78% (dried blood) of peptides display an inter-day CV <30%. Lower limits of detection and quantification were determined for peptides and proteins observed in each biofluid and workflow. Based on each protein and peptide's analytical performance, we could describe the observable, reliable, reproducible, and quantifiable proteomes for each biofluid and workflow. The standardized workflows established here allows for reproducible and quantifiable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow should simplify discovery approaches and facilitate the translation of candidate markers into clinical use.

Application of Homogeneous Liquid-Liquid Microextraction With Switchable Hydrophilicity Solvents to the Determination of MDMA, MDA and NBOMes in Postmortem Blood Samples.

Synthetic drugs for recreational purposes are in constant evolution, and their consumption promotes a significant increase in intoxication cases, resulting in damaging public health. The development of analytical methodologies to confirm the consumption of illicit drugs in biological matrices is required for the control of these substances. This work exploited the development of an extraction method based on homogenous liquid-liquid microextraction with switchable hydrophilicity solvent (SHS) as extraction phase for the determination of the synthetic drugs 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine and N-methoxybenzyl-methoxyphenylethylamine derivates (25B, 25C and 25I) in postmortem blood, followed by liquid chromatography coupled to mass spectrometry in tandem. The optimized sample preparation conditions consisted of using 250 µL of ZnSO4 10% and 50 µL of NaOH 1 mol/L in the protein precipitation step; N,N-dimethylcyclohexylamine was used as SHS, 650 μL of a mixture of SHS:HCl 6 mol/L (1:1 v/v), 500 μL of whole blood, 500 μL of NaOH 10 mol/L and 1 min of extraction time. The proposed method was validated, providing determination coefficients higher than 0.99 for all analytes; limit of detection and limit of quantitation ranged from 0.1 to 10 ng/mL; intra-run precision from 2.16% to 9.19%; inter-run precision from 2.39% to 9.59%; bias from 93.57% to 115.71% and matrix effects from 28.94% to 51.54%. The developed method was successfully applied to four authentic postmortem blood samples from synthetic drugs users, and it was found to be reliable with good selectivity.

The crux of C1-INH testing in everyday lab work

BackgroundThe determination of functional C1-INH is complex and depends on methodology, sample transport, and storage conditions. In clinical practice, we encounter individuals with pathological values which then cannot be proved true, and HAE patients in whom the values were wrongly found to be normal under non-optimum conditions. We aimed to test realistic real-life sample processing conditions for accurate C1-INH determination. MethodsWe conducted two national inter-laboratory comparisons with optimal sample preparation but different dispatch conditions. We also investigated variations of temperature and time, and their influence on C1-INH. ResultsC1-INH levels showed a significantly wider dispersion under suboptimal transport conditions than under optimal conditions (p < 0.00001). Two putatively healthy patient samples turned out to be pathological. Contrary to our expectations, we found no significant trend in a specific direction when the variables of temperature, time and sample material were combined and varied under realistic conditions. However, the range of variation in [%] functionality was markedly greater in supposedly healthy volunteers. Thus, under experimental conditions we obtained false pathological results that were not far from reality. ConclusionC1- INH determination is crucial for the diagnosis of HAE. Time, temperature, and sample handling have a significant impact on this laboratory value, sometimes leading to incorrect values, inaccurate diagnoses, and inappropriate therapies. This underlines the importance of proper handling of samples. If a patient has ambiguous C1-INH values despite optimized conditions, thus hindering a conclusive diagnosis of HAE, we recommend genetic testing.