The reliability and accuracy of the sample results in clinical laboratory tests are influenced by many factors, such as the preanalytical factor, sampling, transportation, storage of samples, as well as the presence of interfering substances in the biomaterial samples. Interfering substances are one of the most common errors in clinical laboratory measurements. The presence of these substances in the samples leads to distortion of the measurement results and endangers the life and health of the patient.

Interference can be caused by the presence of both an endogenous and exogenous substance in the biomaterial sample. The main endogenous interfering factors include: metabolites from pathological conditions such as diabetes mellitus, multiple myeloma, cholestatic hepatitis, haemolysis, i.e. destruction of erythrocytes with the release into the liquid part of the blood of a number of intracellular components (haemoglobin, LDH, potassium, magnesium, etc.), which changes the true results of the determination of the concentration/activity of blood components such as bilirubin, lipase, lipemia, and which alters the results of a number of colorimetric and nephelometric research methods (especially in research into phosphorus, total bilirubin, uric acid, total protein, electrolytes). Exogenous factors include: medicinal products and their metabolites, parenteral nutrition, plasma extenders, anticoagulants; substances taken by the patient, such as alcohol, drugs, food additives, etc.; substances added during the sample preparation, such as anticoagulants, preservatives, stabilizers. Interfering substances distort the results of clinical laboratory measurements by means of the following mechanisms of influence on the analytical system [1,3]:

• By chemical artefact, the interferent can suppress the reaction by competing for reagents or inhibiting the reaction, it can also change the analyte form by complexation or precipitation;
• By distorting the measured parameters, the interfering agent may have properties similar to the analyte, such as fluorescence, colour, light scattering, elution position or electrode response, which are detected and measured;
• By physical artefact exposure to an interfering agent may alter the physical property of the sample matrix, i. e., viscosity, surface tension, turbidity or ionic strength, causing visible changes in the concentration of the measured value;
• By inhibiting enzymes, the interferent can change the activity of the enzyme (analyte or reagent) by sequestering metallic activators, binding to a catalytic centre or oxidizing basic sulfhydryl groups;
• As a mechanism of non-specificity, the interferent can react in the same way as the analyte.
• By means of cross reactivity: an interferent similar in structure to the antigen can cross-react with the antigen in the immunochemical measurement method;

Currently, the international organization CLSI offers only two methods for assessing the effects of potentially interfering substances. According to the first method, the assessment is made by adding potentially interfering substances to the sample of the biomaterial. According to this method, it is necessary to obtain several samples of the relevant biomaterial from healthy people who do not have the interferent of interest. If suitable fresh samples are not available, frozen or lyophilized samples may be used. The selected biomaterial is divided into two parts: a solution with a high concentration of interferent is added to the first part, and a solvent equal in volume to the first solution is added to the second part. For all solutions, the concentration of the measurand is determined and the obtained data are statistically processed. It should be noted that in order to achieve accurate results, all samples are analysed in one measurement cycle, this is required in order to avoid the appearance of other variables, such as calibration or measurement of a batch of reagents. This technique allows the relationship to be determined between the concentration of the interferent and the amount of interference at any concentration of interfering substances within the selected range [1,2].

The second method is to estimate the displacement of individual representative samples of patients compared to highly specific ones. It has low sensitivity to interfering substances, so it is used to establish the true values of the influence of potentially interfering substances. The method is based on a comparative analysis of two methods for determining the measurand: the reference method, or another procedure, which has high precision and selectivity, and the method used by the manufacturer. The analysis is carried out using two groups of patient samples ‒ the test and control samples. Biomaterial samples of the test group of patients should contain one or more potentially interfering substances. This group is selected based on the diagnoses or treatment methods for patients. Control samples are taken from healthy patients whose biomaterial does not contain potentially interfering substances. Samples of the biomaterial obtained from two groups of patients should cover the same concentration range of measurand. The samples obtained are analyzed in two repeats using each measurement procedure. According to the results, statistical processing of the data is carried out and a dot diagram of the comparison of the two studied groups is constructed. The results of the test group of patients are compared with the data of the control group and evaluated for the presence of systematic differences, variability of results or increase in the number of extreme values for the test group. Substances that exhibit a clinically significant effect are considered to be interferents for which the relationship between the interferent concentration and the degree of interference is then evaluated. If no clinically significant effect is observed, the shift, if any, caused by the substance is irrelevant and no further testing is performed. [2].

Thus, in order to achieve the accurate and correct results in clinical laboratory tests, a detailed study of each potentially interfering substance is required, which includes the study of the nature of the substance, the mechanism of influence on the measurand the correct selection of the method of assessment of the influence of the interferent.

List of references

1. Interference Testing in Clinical Chemistry, 3rd Edition — EP07 Paperback — 2018, by NCCLS.
2. Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline — Second Edition. NCCLS document EP9-A2 (ISBN 1-56238-472-4). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2002.
3. Analytical Interference More than Just a Laboratory Problem. Steven C. Kazmierczak, PhD, and Paul G. Catrou, MD, 2000 Jan;113(1):9-11.