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2 March 2026

Classical Effective Techniques to Evaluate Biological Compounds and Materials Toxicity Using Red Blood Cells as Biosensors

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1
Departamento de Microbiología e Inmunología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, San Nicolas de los Garza 66455, Nuevo León, Mexico
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Facultad de Medicina y Ciencias Biomédicas, Universidad Autónoma de Chihuahua, Chihuahua 31125, Chihuahua, Mexico
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Universidad Tecnológica de México (UNITEC), Campus Monterrey, San Nicolas de los Garza 66499, Nuevo León, Mexico
4
Facultad de Zootecnia y Ecología, Universidad Autónoma de Chihuahua, Chihuahua 31453, Chihuahua, Mexico
Chemosensors2026, 14(3), 55;https://doi.org/10.3390/chemosensors14030055
This article belongs to the Section (Bio)chemical Sensing
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Abstract

Red blood cells represent a widely used cellular model in cytotoxicity studies, particularly in hemocompatibility assessments. As enucleated cells, which are abundant and easily accessible in both humans and animals, red blood cells allow for rapid, reproducible, and low-cost evaluation of the toxicity of bioactive compounds, whether natural, synthetic, or nanoparticulate. From a functional perspective, the red blood cell membrane is highly sensitive to physical and chemical environmental changes (osmolarity, temperature, pH, and the presence of oxidizing agents). This sensitivity makes red blood cells an effective biosensor for detecting membrane damage, hemolysis, oxidative stress, methemoglobin formation, and aggregation processes. Therefore, in vitro tests using red blood cells allow for the preliminary evaluation in preclinical development, particularly for the early screening of cytotoxicity, membrane-disruptive effects, and hemocompatibility of small molecules, nanomaterials, and blood-contacting biomaterials. These techniques include hemocompatibility tests, evaluation of oxidative and osmotic damage, and evaluation of erythrocyte aggregation and function. However, the use of red blood cells as a cytotoxicity model also has significant limitations. As anucleate cells, erythrocytes lack organelles such as nuclei, mitochondria, or lysosomes, which prevents the evaluation of their effects on key intracellular processes such as protein synthesis, cell signaling, apoptosis, or endoplasmic reticulum stress. This lack of cellular complexity limits their usefulness as a sole model in studies of systemic toxicity or tissue-specific cytotoxicity. These tools offer an effective preliminary approach to anticipating risks in biomedical and pharmacological research.

1. Introduction

The ongoing development of drugs, nanoparticles, natural formulations, medical devices, and other therapeutic agents is one of the main strategies for disease control and treatment worldwide [1,2]. However, factors such as the growing resistance to conventional treatments, biological variability, and the limited availability of effective medications require the design of new therapeutic alternatives that are not only effective but also safe for the body [3].
Despite the therapeutic potential offered by various natural and synthetic sources, it is essential to evaluate their biocompatibility, especially in compounds that interact with the circulatory system [4]. In this context, compatibility with erythrocytes becomes particularly relevant, since these abundant, enucleated cells are highly sensitive to physical and chemical effects [5]. Exposure to bioactive compounds induces hemolysis, hemoprotection, membrane oxidation, osmotic fragility, or abnormal aggregation, which may compromise erythrocyte functionality and trigger systemic adverse effects [6].
Various in vitro and in vivo tests have been implemented to evaluate the compatibility of organic and inorganic compounds and even medical devices, in order to minimize future side effects, when implemented in humans or animals [2,7]. However, many of the current biocompatible evaluation techniques require sophisticated infrastructure or involve high costs, which hinders their routine use in laboratories with limited resources [8].
The evaluation of biocompatibility, as well as the selective toxicity of nanostructured compounds, biomaterials, and bioactive compounds, represents a critical stage during the early phases of pharmacological development with biomedical impact. Initially, the interaction of these materials with easily accessible and rapidly obtainable biological models, such as erythrocytes, is particularly relevant, as red blood cells are highly sensitive to physicochemical alterations resulting from direct contact with tested compounds. Therefore, erythrocytes have been widely employed as a simple, accessible, and physiologically relevant model for the preliminary screening of compound selectivity and toxicity, including nanocomposites [9,10].
In recent years, several studies have demonstrated the importance of erythrocytes as cytotoxicity models, highlighting their role in the development of biosensors for membrane damage, oxidative imbalance, and hemolytic activity. Nevertheless, most of these studies focus on specific classes of materials, such as nanoparticles, implantable biomaterials, or molecular mechanisms, rather than providing a comprehensive methodological perspective of the diverse assays that can be integrally implemented in a preliminary analytical workflow [11,12,13].
Despite the widespread use of classical assays, such as hemolysis, oxidative damage, osmotic fragility, erythrocyte aggregation, and methemoglobin formation, experimental variations, interpretation thresholds, and reporting criteria across different studies continue to represent a major comparative challenge. This lack of methodological standardization has been recognized as an important limitation for inter-study comparison and result reproducibility [14,15,16]. In parallel, recent reviews focusing on antioxidant activity or assay optimization emphasize the continued relevance of erythrocyte-based models [17].
In this scenario, classical hemocompatibility techniques based on human or animal erythrocytes remain highly accessible, sensitive, and reproducible tools [11,18]. These tests allow for the early identification of potential toxic effects and may serve as, or be considered as, an effective, economically reproducible, and low-cost preliminary step before advancing to more complex studies.
Therefore, the present review was considered necessary to consolidate, update, and critically analyze the classical erythrocyte-based methods that are currently employed to evaluate the toxicity and hemocompatibility of biological compounds and materials. Unlike previous reviews, this work integrates the experimental basis, biological relevance, advantages, and limitations of each assay, while also proposing practical interpretation criteria to facilitate comparison across studies. In doing so, this review aims to provide researchers with a comprehensive and practical reference for the rational selection of erythrocyte-based assays as a preliminary screening tool prior to advancing to more complex in vitro or in vivo models.

2. Erythrocyte Hemocompatibility

2.1. Biophysical Properties of the Erythrocyte Membrane

The erythrocyte membrane is crucial for erythrocyte compatibility, as it is the barrier that separates intracellular content from the extracellular environment. Its structure and biophysical properties largely determine the response of erythrocytes to exposure to compounds [10]. The lipid membrane, composed mainly of phospholipids and cholesterol, is involved in selective permeability and in maintaining the biconcave shape of erythrocytes, which is essential for their flexibility and functionality [19].

2.2. Structure and Properties of the Erythrocyte Membrane

The red blood cell membrane is organized into a lipid bilayer that is anchored to an underlying protein network known as the cytoskeleton, composed primarily of spectrin and actin [20]. This structure gives red blood cells their flexibility, allowing them to navigate through the narrowest capillaries and preventing their rupture [21]. The membrane also contains several integral proteins, such as the anion channel, the chloride–bicarbonate transporter, and various glycoproteins, which are essential for pH regulation and cell integrity [22,23].

2.3. Interactions with External Compounds

Exposure of erythrocytes to bioactive compounds can induce significant alterations in membrane stability and cellular homeostasis. These interactions may promote oxidative stress through the excessive generation of reactive oxygen species (ROS), which play a central role in erythrocyte damage. Due to their high polyunsaturated fatty acid content, erythrocyte membranes are particularly susceptible to oxidative attack, making them a sensitive model for toxicological assessment. ROS can initiate lipid peroxidation processes by targeting bilayer phospholipids, as well as oxidizing cytoskeletal proteins and hemoglobin, leading to structural and functional impairment of the erythrocyte membrane [24]. Oxidative modification of membrane proteins and lipids disrupts membrane fluidity, deformability, and permeability, which compromises the mechanical stability of erythrocytes during circulation. As a consequence, these alterations can increase membrane fragility, ultimately resulting in hemolysis and the subsequent release of hemoglobin into the plasma [25]. Therefore, erythrocyte-based assays provide a valuable and sensitive approach for evaluating the cytotoxic and pro-oxidant effects of bioactive compounds and biomaterials, as well as their potential to induce membrane damage under physiological conditions.
On the other hand, exposure of erythrocytes to bioactive compounds can induce membrane damage through multiple pathways, which largely depend on the chemical structure, physicochemical properties, and redox behavior of the compound. Different classes of bioactive compounds trigger membrane injury through distinct mechanisms, including direct interaction with the lipid bilayer, metal-catalyzed redox reactions, hemoglobin modification, or disruption of cytoskeletal proteins [9,10].
Amphiphilic and surface-active compounds, such as surfactants, saponins, and antimicrobial peptides, interact with erythrocytes primarily through direct insertion into the lipid bilayer. Their amphipathic structure facilitates partitioning into membrane lipids, thereby altering lipid packing, membrane fluidity, and surface tension. This interaction promotes pore formation and osmotic lysis, processes that frequently occur independently of oxidative stress. The hemolytic activity of these compounds correlates with hydrophobic chain length, charge density, and overall amphiphilic balance [26,27].
Metal ions and metal-based nanoparticles induce erythrocyte damage predominantly via catalytic redox mechanisms. Transition metals such as iron, copper, and silver promote Fenton or Fenton-like reactions, generating highly reactive hydroxyl radicals at the membrane surface. These reactive species initiate localized lipid peroxidation and protein oxidation, making membrane damage more dependent on oxidation state, coordination chemistry, and surface reactivity than on strictly dose-dependent toxicity [12,13].
Redox-active organic compounds, including quinones and related conjugated systems, exert their toxicity through redox cycling mechanisms. These compounds undergo repeated reduction–oxidation reactions, resulting in sustained generation of reactive oxygen species and rapid depletion of erythrocyte antioxidant defenses. Structural features such as conjugated ring systems, electron-withdrawing substituents, and redox potential determine their pro-oxidant efficiency and the extent of both membrane and hemoglobin oxidation [28,29].
Hemoglobin-reactive agents, such as phenylhydrazines and nitrite-derived compounds, primarily target intracellular hemoglobin rather than membrane lipids. These substances induce hemoglobin denaturation, methemoglobin formation, and Heinz body aggregation. Secondary interactions between damaged hemoglobin and cytoskeletal proteins reduce erythrocyte deformability and promote premature hemolysis, with membrane rupture occurring as a downstream consequence of intracellular protein damage [30,31].
Polyene antibiotics and other sterol-binding agents interact selectively with membrane sterols, forming transmembrane pores that disrupt ionic balance and membrane integrity. Their hemolytic activity depends on structural complementarity between the polyene macrolide backbone and cholesterol, underscoring the importance of lipid composition and molecular recognition in erythrocyte susceptibility [12].
Finally, compounds that target the cytoskeleton compromise erythrocyte integrity by modifying spectrin–actin interactions or associated anchoring proteins. Oxidative modification or covalent binding to cytoskeletal components reduces membrane elasticity and mechanical stability, increasing sensitivity to shear stress and promoting mechanical hemolysis during circulation [32,33].

2.4. Oxidative Stress and Activation of Cellular Pathways

Oxidative stress is one of the most common molecular responses in erythrocytes exposed to potentially toxic compounds. When erythrocytes are exposed to an environment with a high concentration of reactive oxygen species (ROS), endogenous antioxidants such as glutathione peroxidase and superoxide dismutase are involved in neutralizing these species [34,35]. However, if the amount of ROS exceeds the antioxidant activity, lipoperoxidation of membrane lipids may occur, leading to alterations in the fluidity and stability of the lipid bilayer, and ultimately, cell lysis [36].
In addition, some compounds induce the activation of immunological pathways, such as complement activation, which is triggered by the formation of immune complexes on the surface of erythrocytes, promoting opsonization and facilitating phagocytosis [37,38]. Although this activation is more relevant in the context of extrinsic materials, certain drugs or nanoparticles also induce adverse immunological reactions [39,40].
In addition to oxidative damage, some compounds induce erythrocyte injury through activation of immunological pathways, particularly the complement system. Complement activation may be triggered by antibody–antigen complexes or foreign surfaces interacting with erythrocyte membrane proteins, leading to opsonization and formation of the membrane attack complex (MAC), which can cause intravascular hemolysis or promote extravascular clearance by phagocytes [41,42].
Another important pathway involves dysregulation of intracellular calcium (Ca2+) homeostasis. Oxidative stress or membrane-interacting compounds can increase Ca2+ permeability, resulting in elevated intracellular Ca2+. This influx activates Ca2+-dependent enzymes and disturbs membrane phospholipid asymmetry, ultimately reducing erythrocyte deformability and increasing susceptibility to damage. Elevation of intracellular Ca2+ is also a major driver of eryptosis, a form of programmed erythrocyte death characterized by cell shrinkage, membrane blebbing, and phosphatidylserine exposure, which promotes macrophage recognition and clearance independent of direct hemolysis [43].

2.5. Erythrocyte Aggregation

Red blood cells aggregate in response to alterations in the physicochemical properties of blood. This may occur through interactions between the glycocalyx of red blood cells, or through changes in the membrane surface charge caused by certain compounds [44]. Abnormal red blood cell aggregations affect blood viscosity and alter microvessel flow, compromising blood circulation and promoting thrombus formation [45].

2.6. Molecular Considerations on Hemocompatibility

The compatibility of a compound with erythrocytes depends on its structural characteristics and its physicochemical properties, which include its charge, size, and shape [13]. Compounds with a negative or neutral charge are usually better tolerated by erythrocytes, whereas compounds with positive charges induce greater electrostatic interaction with the membrane that triggers hemolysis or oxidative stress [46,47]. Similarly, the particle size (in the case of nanoparticles) may influence the potential of the compound to induce erythrocyte aggregation or mechanical hemolysis [12].

3. Critical Aspects and Sources of Variability in Erythrocyte Preparation for Hemocompatibility Assays

Proper red blood cell preparation represents a major source of variability in hemocompatibility assays and directly affects the reliability and interpretation of experimental outcomes [14,48]. The preparation steps summarized below are representative of commonly reported practices and are discussed in terms of their potential influence on assay reproducibility when preparing human or animal red blood cells:
  • Blood collection: Fresh blood is obtained from human or animal donors, following ethical and safety regulations. The blood must be treated with an anticoagulant, such as EDTA (ethylenediaminetetraacetic acid) or citrate, to prevent clotting [14].
  • Centrifugation: The blood sample is centrifuged at 3000 rpm for 10 min to separate the cellular components from the blood. The plasma is carefully removed, leaving the red blood cells at the bottom of the tube [49].
  • Red blood cells washing: Red blood cells are washed three times with isotonic saline, such as 0.9% NaCl or phosphate-buffered saline (PBS), to remove plasma residues. Centrifugation is performed at 3000 rpm for 5 min between each wash [50]. Proper red blood cell preparation represents a major source of variability in hemocompatibility assays and directly affects the reliability and interpretation of experimental outcomes [51].
  • Red blood cell suspension: After washing, red blood cells are suspended in an isotonic solution (0.9% NaCl or PBS pH 7.2, depending on the assay) at a concentration of approximately 2% to 5% by volume [50].
Proper red blood cell preparation is a critical factor influencing the reproducibility of hemocompatibility assays and the interpretation of their results. Variations in sample handling, washing procedures, and experimental conditions have been widely reported to introduce significant variability, potentially leading to inconsistent or misleading outcomes across studies [52].

3.1. Hemocompatibility Tests

Classical hemocompatibility tests are classified according to the type of red blood cell damage being assessed. The parameters for these assays are summarized in Table 1. The tests differ in the mechanism of injury they detect and therefore provide complementary information [53]. Hemolysis-based assays are direct indicators of membrane disruption but do not identify the underlying cause of damage. In contrast, oxidative assays (H2O2 exposure, TBARS, and FOX) are more sensitive to early biochemical alterations and allow detection of subhemolytic oxidative stress. Osmotic fragility tests primarily reflect structural membrane stability under physicochemical stress, whereas aggregation assays provide hemorheological information not captured by lysis-based methods. Methemoglobin formation assesses functional impairment of hemoglobin and complements structural endpoints. Together, these approaches vary in sensitivity, specificity, and physiological relevance, supporting their combined use for a comprehensive evaluation of hemocompatibility.
In addition to mechanistic differences, the assays also vary in their experimental parameters. Most hemolysis and oxidative tests share similar incubation temperatures (typically 37 °C) and spectrophotometric detection near 540 nm, enabling comparable quantification of hemoglobin release. However, oxidative assays require broader reaction conditions and, in the case of TBARS, higher temperatures and longer centrifugation steps, reflecting their chemical detection principles. Functional and aggregation tests differ in detection wavelength and handling conditions, indicating that assay selection depends not only on the type of erythrocyte damage assessed but also on analytical sensitivity, processing requirements, and experimental complexity. Collectively, the parameters compiled in Table 1 provide a structured framework for selecting appropriate assays and for comparing methodological requirements across studies.

3.1.1. Assessment of Hemolysis and Membrane Damage

  • Direct hemolysis: Prepared red blood cells are exposed to the compound at various concentrations and incubated at 37 °C for oxidation. The release of hemoglobin into the supernatant is then measured by spectrophotometry at 540 nm. An increase in absorbance indicates hemolysis [54].
  • Heat-induced hemolytic activity: Red blood cells are exposed to specific temperatures (e.g., 50 °C to 60 °C) for 30 min, after which hemolysis is measured through the release of hemoglobin [55].
  • Cold-induced hemolytic activity: Red blood cells are incubated at low temperatures (0 °C to 4 °C) for 30 min, and hemolysis is then measured. This type of test assesses the stability of red blood cells to extreme temperature changes [55].

3.1.2. Evaluation of Oxidative Damage

  • Antihemolytic activity by oxidative damage induced by H2O2: Red blood cells are exposed to various concentrations of H2O2 to induce oxidative stress. The compound under study is added to the red blood cells suspension before incubation. After a predetermined time (e.g., 30 min), hemolysis is spectrophotometrically measured. The antioxidant activity of the compound is assessed by its potential to reduce hemolysis [56].
  • Hemoprotection activity by 2,2′-azobis (2-methylpropionamide)-dihydrochloride (AAPH): Red blood cells are incubated with APPH to generate free radicals. The induced hemolysis is then measured and the ability of a compound to protect red blood cells from damage caused by APPH-generated free radicals is assessed [54].
  • Thiobarbituric acid reactive substances (TBARS) test: Erythrocytes are incubated with an oxidizing agent, such as H2O2, to induce lipid peroxidation. Thiobarbituric acid is then added and heated under commonly reported conditions (e.g., ~95 °C for 30 min), depending on the specific protocol used. After cooling, absorbance at 532 nm is measured to evaluate lipid peroxidation products, such as malondialdehyde, indicating oxidative damage to erythrocyte membranes [56].
  • Xylenol orange test: Red blood cells are incubated with an oxidizing agent such as H2O2 to induce lipid peroxidation. Xylenol orange reagent is then added, which binds to lipid peroxidation products. The mixture is incubated for a specified time, and the absorbance at 540 nm is measured, indicating the level of oxidative damage to the red blood cell membranes [57].

3.1.3. Osmotic Damage Assessment

  • Antihemolytic activity by osmotic damage (hypotonicity): Red blood cells are exposed to NaCl solutions with decreasing concentrations (from 0.9% to 0.1% NaCl). Hemolysis is then spectrophotometrically measured, and the potential of the compound to protect erythrocytes from osmotic hemolysis is assessed [55].

3.1.4. Evaluation of Erythrocyte Aggregation and Function

  • Albumin-induced antiaggregatory activity of erythrocytes: Red blood cells are incubated with albumin at various concentrations to induce aggregation. Red blood cell aggregation is then measured using dark-field microscopy or viscosity techniques, and the potential of the compounds to reduce aggregation is assessed [58].
  • Measurement of methemoglobin in human erythrocytes: Red blood cells are incubated with oxidizing agents to induce methemoglobin formation. The methemoglobin concentration in red blood cells is then measured by spectrophotometry at 630 nm [59].
  • Anti-methemoglobin activity: The potential of a compound to prevent or reverse the formation of methemoglobin is assessed. Red blood cells are exposed to an oxidation source (NaNO3), and the amount of methemoglobin in the presence of the protective compound is measured [31,59,60,61].
Collectively, these hemocompatibility assays provide sensitive and early indicators of cytotoxicity and biocompatibility for a wide range of materials, including small molecules, nanomaterials, and blood-contacting biomaterials. Because erythrocytes lack nuclei and repair mechanisms, alterations in membrane integrity, oxidative balance, or deformability directly reflect physicochemical interactions at the cell surface. Consequently, these tests are particularly valuable for identifying sublethal or early-stage damage that may not be detected in more complex cellular models, supporting their use as first-line screening tools in toxicological and biocompatibility assessments.
Table 1. Hemocompatibility test parameters.
Table 1. Hemocompatibility test parameters.
GroupTest°Cλ (nm)Incubation TimeCentrifugationVolume (μL)Positive ControlNegative ControlReference
HemolysisInduced hemolysis375401–2 h3000 rpm, 5 min200–500Distilled waterIsotonic saline solution[54,55,62,63]
Heat-induced hemolysis50–6054030 min3000 rpm, 5 min200–500Distilled water at 60 °CPBS at 37 °C[55]
Cold-induced hemolysis0–454030 min3000 rpm, 5 min200–500Thermal shock (quick-freezing)PBS at 37 °C[55]
OxidativeOxidative damage by H2O23754030–60 min3000 rpm, 5 min200–500H2O2 (100–500 μM)Red blood cells in PBS[56]
Hemoprotection by APPH3754060–120 min3000 rpm, 5 min200–500APPHPBS or known protective compound[64]
TBARS9553260 min3500 rpm, 10 min200–300Fe2+ + ascorbic acidOxidizer-free PBS[56]
Xylenol orange (FOX)RT of 3754030–60 min3000 rpm, 5 min200–300H2O2 or oxidized lipidsPeroxide-free solution[57]
OsmoticOsmotic damage3754030–60 min3000 rpm, 5 min200–500Hypotonic solution (0.1–0.3% NaCl)Isotonic saline solution (0.9% NaCl)[55]
AggregationAlbumin antiaggregant3740X30–60 minMild agitation200–500Albumin (high concentration)Untreated erythrocytes[58]
FunctionMethemoglobin3763030 min3000 rpm, 5 min200–500NaNO2 (1 mM)Red blood cells in PBS[59]
Anti-methemoglobin3763030–60 min3000 rpm, 5 min200–500NaNO2 + control compoundNaNO2[65]
Table 2 presents various erythrocyte-based assays that allow for the specific evaluation of cellular damage caused by bioactive compounds or materials in contact with blood. Induced, thermal, and cold hemolysis tests help detect alterations in the erythrocyte membrane under different conditions. A value equal to or below 5% is associated with compatibility, whereas higher percentages indicate progressive damage, ranging from moderate to severe. Induced hemolysis is a crucial parameter in determining the biological activity of natural extracts. Due to the lack of clear criteria in the literature to determine whether a natural extract exhibits hemolytic activity, we propose the classification outlined in Table 3.
Table 2. Parameters for the interpretation of classical erythrocyte-based assays for the toxicological evaluation of bioactive compounds and biomaterials.
Table 3. Interpretation of hemolytic IC50 values for natural extracts.
On the other hand, oxidative assays include peroxide-induced models (H2O2, AAPH) and methods that quantify lipid peroxidation products (TBARS, FOX). Compounds that keep damage levels below 10% or 0.1 nmol/mL are considered protective, while intermediate or high values indicate oxidative effects [10,17,35,56]. These tests allow for classification of compounds based on their ability to mitigate or induce oxidative stress at the cellular level.
The osmotic damage test evaluates erythrocyte sensitivity in hypotonic solutions. Integrity is maintained with less than 5% lysis, while higher percentages reflect disruption of cellular homeostasis. In parallel, the albumin-induced aggregation test identifies whether a compound promotes or prevents cellular agglutination, with thresholds that help determine pro- or anti-aggregating effects.
Regarding hemoglobin functionality, assays for methemoglobin formation and reversion determine the degree of oxidation of the heme group. Low values indicate a protective effect, while lack of reversion reflects a compound’s inability to counteract the damage. This information is key to distinguishing between adverse effects and potentially therapeutic activities.
In this context, the protective or antihemolytic ability of a compound is assessed by its capacity to reduce hemoglobin release compared with oxidant-treated controls. This effect is typically expressed as a percentage of hemolysis inhibition and allows the calculation of concentration–response relationships, from which IC50 values can be derived. Therefore, the IC50 values reported in Table 3 reflect the concentration required to reduce hemolysis by 50% under the specified oxidative conditions, providing a quantitative measure of erythrocyte protection.

4. Application of Erythrocyte-Based Hemocompatibility Tests

Table 4 summarizes the main types of compounds and materials that have been evaluated using hemocompatibility-related assays. A wide variety of compounds have been evaluated under thermal stress conditions to determine their influence on erythrocyte membrane stability. In these assays, red blood cells are subjected to controlled heat exposure to induce membrane destabilization, and test compounds are assessed for their ability to reduce temperature-induced hemolysis. Thus, the readout reflects whether a compound exacerbates membrane damage or confers a protective effect against thermally induced stress, rather than directly alleviating heat stress itself [10]. For example, extracts of Ibervillea sonorae and Scenedesmus sp. have been evaluated against murine lymphoma cells (L5178Y-R), while silver–chitosan nanoparticles have been analyzed for their antimicrobial activity and hemocompatibility. Liposomal delivery systems with endophytic extracts have also been explored in glioblastoma models. Polymers such as chitosan, PEG (polyethylene glycol), and PLGA (polylactic-co-glycolic acid) are frequently studied in combination with bioactive compounds for topical or biomedical applications. In addition, cosmetic formulations and adjuvants have been assessed through oxidative stress and aggregation assays to predict their irritant or immunostimulatory potential. Finally, materials intended for biomedical devices, such as catheters or sensors, are commonly evaluated through contact hemolysis and thermal compatibility tests. These approaches highlight the importance of early biocompatibility assessments in the development of new therapeutic or diagnostic platforms.
Table 4. Application of erythrocyte-based hemocompatibility tests.

5. Discussion

The search for safe and effective bioactive compounds in areas such as pharmacognosy, functional foods, and nanomaterials requires efficient experimental models that highlight accessibility, sensitivity, and physiological relevance to estimate the biocompatibility of nanomaterials, as well as bioactive compounds, natural or synthetic [84,85]. From this context, a stable, reliable, and representative scaffold of the behavior of cell membranes when exposed to physical or chemical agents is essential [10]. Based on this, classical assays with red blood cells provide critical information on membrane stability and allow the evaluation of the potential hematic cytotoxicity of natural extracts, secondary metabolites, or nanostructured systems [16]. These tests, by quantifying the release of hemoglobin or evaluating the protection against oxidative, osmotic or thermal damage, allow an early classification of the potential risk of the compounds. Therefore, their usefulness is consolidated as an initial screening tool in the rational design of bioactive products [9].
In pharmacognosy, extracts derived from plants, fungi, and endophytic microorganisms present diverse chemical structures, whose interactions with the cell membrane are difficult to predict during the early stages and subsequent stages of the analysis of crude extracts, partitions, and fractions of the bioactive extracts [86]. Assays such as the H2O2 antihemolytic test or osmotic fragility are especially useful for identifying compounds with antioxidant or membrane-stabilizing properties, which is desirable not only to reduce toxicity, but also as an indication of possible therapeutic benefits by directing studies from early stages and directing subsequent studies of bioactive extracts and compounds [17].
For functional and nutraceutical ingredients, red blood cells also allow for safety assessment in complex matrices prior to the use of animal models. Detection of subhemolytic or aggregation effects alert us to undesirable interactions that compromise the biosafety of these products. Thus, these tests align with the 3R principles (reduce, refine, and replace) by reducing the use of animal models in preliminary phases [15].
In the field of nanomaterials, such as liposomes, metallic nanoparticles or biofunctional polymers, red blood cells estimate the hematic compatibility of the transport systems [12]. Excessive hemolysis may indicate an unstable surface or an inadequate surface charge [87]. Moreover, the capacity of certain nanostructured compounds to protect or alter the erythrocyte membrane can be correlated with their behavior in blood and predict adverse events such as thrombogenicity or systemic cytotoxicity [88].
It is important to emphasize that, although erythrocytes represent a relatively simple experimental model, their high sensitivity allows for the detection of early adverse effects that may not be evident in more resistant or complex cell systems. This makes them an effective preliminary screening tool prior to scaling up studies to more advanced in vitro models, such as tumor cell lines, or to in vivo animal models [88]. Moreover, clearly defined interpretation criteria for each erythrocyte-based assay, together with rigorously established positive and negative controls, enable a systematic and reliable comparison of the hemolytic profiles of different compounds or materials. Consequently, these methodologies facilitate the early assessment of toxicity and blood compatibility and are particularly valuable during the initial stages of the development of new therapeutic agents or biomaterials, as well as in screening assays [80].
Given these advantages, future studies should aim to further standardize erythrocyte-based assays and expand their use as early screening tools for hemocompatibility and toxicity. Improving methodological consistency, incorporating quantitative endpoints, and validating results across different types of bioactive compounds and nanomaterials will strengthen their predictive value. Emerging analytical approaches, including automated image-based analysis and data-driven tools, may enable the detection of subtle erythrocyte morphological changes beyond conventional hemoglobin release measurements. In pharmacognosy and nutraceutical research, integrating erythrocyte assays with metabolomic profiling could further clarify how specific bioactive fractions interact with and stabilize membrane structures at the molecular level. Additionally, combining red blood cell assays with complementary in vitro models may provide a more comprehensive evaluation of blood compatibility. Together, these advances are expected to enhance the reliability and translational relevance of erythrocyte-based testing and support its broader application in the development of safe therapeutic, nutritional, and nanotechnology-related products.
In conclusion, the use of red blood cells as a biocompatibility model represents an efficient, cost-effective, and reproducible strategy for the preliminary toxicological evaluation of bioactive compounds. Its broad applicability in pharmacognosy, functional ingredient design, and nanomaterial development positions erythrocyte-based assays as a critical step toward the development of safe products with therapeutic or nutritional potential. Beyond their current use, the re-evaluation and further standardization of these assays, together with the incorporation of quantitative endpoints, automation, and complementary in vitro and in silico approaches, may substantially enhance their predictive capacity. Although erythrocyte-based assays have been widely applied in hemocompatibility and toxicity testing, their integration into predictive or standardized screening frameworks remains limited. This is not due to a lack of biological relevance, but rather to methodological heterogeneity, variability in erythrocyte preparation and assay conditions, and the absence of harmonized acceptance criteria across laboratories. In addition, erythrocytes are often regarded as oversimplified models, which has restricted their broader adoption despite their sensitivity to early membrane and oxidative damage. Addressing these limitations may help optimize early-stage screening strategies, reduce reliance on animal models, and support translational research across areas such as healthcare, food science, and nanotechnology.
Within a comprehensive biocompatibility and toxicity assessment framework, erythrocyte-based assays are best positioned as early-stage screening tools. Their simplicity and sensitivity to membrane and oxidative damage enable rapid identification of potentially harmful compounds. Findings from these assays can subsequently be complemented by more complex in vitro systems, including nucleated blood cells, endothelial models, and immune-based assays, and, when necessary, by ex vivo or in vivo studies. In this tiered strategy, erythrocyte assays do not replace advanced models but support informed decision-making by prioritizing candidates and reducing unnecessary downstream testing.

6. Conclusions

Red blood cell-based hemocompatibility assays provide a sensitive model for the early evaluation of interactions between bioactive compounds, biomaterials, and cell membranes. As discussed in this review, erythrocytes allow the assessment of diverse damage mechanisms, including membrane disruption, oxidative stress, and regulated clearance processes. When key experimental variables are properly controlled, these assays offer meaningful mechanistic insight and predictive value, supporting their use as an initial screening tool in pharmacological and toxicological studies.

Author Contributions

Conceptualization, C.I.R.-S. and N.E.R.-G.; investigation, A.L.D.-M., D.L.C.-P., A.G., B.E.C.-V. and A.R.C.-G.; writing—original draft preparation, C.I.R.-S., N.E.R.-G. and R.G.-F.; writing—review and editing, P.T.-G., C.I.R.-S. and R.G.-F.; supervision, C.M.Q.-F. and R.G.-F.; funding acquisition, C.I.R.-S., N.E.R.-G. and R.G.-F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Consejo Nacional de Humanidades, Ciencia y Tecnología 445572 to C.I.R-S.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study.

Conflicts of Interest

The authors declare no conflicts of interest.

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