Development and validation of a new Spanish instrument to measure health-related quality of life in patients with allergic rhinitis: the ESPRINT questionnaire.

OBJECTIVES: To develop and validate an instrument to measure health-related quality of life (HRQOL) specific to patients with allergic rhinitis (AR) and primarily for use in Spanish and Spanish-speaking populations. METHODS: An initial item pool was generated from literature review, focus groups with AR patients, and consultations with clinical experts. Item reduction was performed using clinimetric and psychometric approaches after administration of the item pool to 400 AR patients. The resulting instrument's internal consistency, test-retest (2-4 weeks) reliability, known groups and convergent validity, and sensitivity to change were tested in a longitudinal, observational, multicenter study in 210 AR patients who also completed the Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ). RESULTS: The new questionnaire took a mean (SD) of 7.1 (5.4) minutes to answer. Floor and ceiling effects were less than 15% on all dimensions. Cronbach's alpha values and intraclass correlation coefficient values for six of the sevendimensions and the overall score exceeded 0.70. Statistically significant differences (P < 0.01) were observed on all ESPRINT-28 dimensions and the overall score between patients with mild (mean overall score 1.97, SD 0.99), moderate (mean overall score 2.78, SD 0.88), and severe AR (mean overall score 3.89, SD 0.87). Patients with persistent AR had worse scores (P < 0.05) on all dimensions than patients with intermittent AR. Correlations between the ESPRINT-28 and the RQLQ were generally as expected. Effect sizes for score changes between the two study visits ranged from 0.96 to 1.76 for individual dimensions and the overall score. CONCLUSIONS: This new, Spanish-developed instrument to measure HRQOL in AR patients has shown good reliability, validity, and sensitivity to change. It has also proved easy to use and administer.

Flow cytometric basophil activation test: a review.

Flow cytometry is a technique enabling the analysis of physical and biological characteristics of cells or other biological particles when labeled with antibodies coupled to fluorochromes or other dyes. The basophil activation test (BAT), also called flow-cytometric allergen stimulation test (FAST) [commercially available under the name of Flow CAST (Bühlmann Laboratories) or BASOTEST (Beckton-Dickinson)] is based on the in vitro allergen-induced specific activation of basophils. This assay rests on the demonstration of some membrane protein markers that appear after exposure to the allergen. This paper reviews some of the literature about the use of this technique in the investigation of immediate-type allergies to inhalant allergens, drugs, and foods, as well as our own experience with this diagnostic technique. Flow cytometry is a reliable method for the quantification of basophil activation after allergenic stimulus in vitro and in vivo. It also enables us to detect allergic and pseudoallergic reactions because of other mechanisms than allergen-specific IgE antibodies. Further clinical evaluation of this technique will allow validation and better establishment of its diagnostic value in allergy.

Identification of a 36-kDa olive-pollen allergen by in vitro and in vivo studies.

BACKGROUND: Ole e 1 has been considered the major allergen of olive (Olea europaea) pollen. Some other relevant allergens (Ole e 2, 3, 4, and 6) have been recently described. This work aimed to study the IgE-binding frequency of a 36-kDa protein from O. europaea pollen in a large population of olive-allergic patients, its allergenic reactivity in vivo, and its presence in olive pollens of different origin, as well as in other relevant allergenic pollens. METHODS: Identification of IgE-binding components from O. europaea pollen extracts was elucidated by inhibition of SDS-PAGE immunoblotting using recombinant profilin (Ole e 2) and Ole e 1 molecules. The IgE-binding frequency of the 36-kDa protein was estimated by Western blot in a sample of 120 sera from olive-allergic patients. The cutaneous test with the 36-kDa protein was performed by intradermoreaction in allergic patients and control subjects. RESULTS: Exactly 83% of the sera from O. europaea-allergic patients recognized a protein with an apparent molecular weight of 36 kDa, under reducing conditions. It was detected by sera from monosensitized and polysensitized patients, showing a higher IgE frequency than the major allergen Ole e 1 (59%) and the minor profilin (Ole e 2) allergen (27%). Similar reactivity rates (79%) was found by intradermal test. Extracts from olive pollens collected in California presented a much higher amount (around 16-fold on average) of the 36-kDa protein than those from pollens of Spanish origin. The presence of similar allergens was detected only in closely related species (Syringa, Fraxinus, Ligustrum), and not in other common allergenic pollens. CONCLUSIONS: The 36-kDa protein constitutes a major allergen for olive-sensitized patients, but it is not equally represented in O. europaea pollens of different origins.

[Bacterial infection in the etiology of bronchial asthma]. / Bakterial'naia infektsiia v étiologii bronkhial'noi astmy.

The role of bacterial infection in etiopathogenesis of bronchial asthma should not be underestimated. The use of maintenance instead of causal treatment is not valid. We insist on antibiotic therapy and long-term bacterial immunotherapy of bronchial asthma and support value of bacterial antigen potentiating the action of inhalation antigen. This is confirmed by our treatment results which are very good.

Bacterial infection as an important triggering factor in bronchial asthma.

It is very surprising that in recent decades, the bacterial infection factor has been so overlooked in the causal treatment of bronchial asthma. Emphasis is put in the viral infection, but the bacterial infection usually associated with it is ignored. In several publications, we have insisted on the importance of the bacterial infection factor in the etiopathogenesis of bronchial asthma. It is alarming that even in the international consensus on its treatment this aspect is overlooked. In the first decades of this century, great importance had already been put on bacterial infection in the triggering of bronchospasm. In this review, we insist on this role of bacterial infection, which comes as a result of our extensive experience in this area, and the fact that in the last 10 years many authors have proven its responsibility at a bronchial mucosa level. In due time, we may be able to prove that the bacterial antigens can potentiate the action of inhalant allergens. Some authors have even proven that the action of these bacterial antigens even more energetically increases the number of intraepithelial dendritic cells in the bronchial mucosa after inhalation of bacterial lipopolysaccharide. Bystander respiratory bacterial infections can also directly modulate T helper 1 and 2 selection parallel to the immune response to inhalant allergens. Recent studies have also proven that in respiratory infection, bacterial antigens hold the main responsibility in the inflammatory and bronchospastic response in the etiopathogenesis of bronchial asthma. Therefore, a consequent treatment of the infection is required, by means of wide spectrum antibiotics, as well as prescription of bacterial immunotherapy, as we have emphasized on other occasions. In conclusion, we must try to cure asthmatic patients and not to maintain them with inhalers and unnecessary corticosteroid therapy, since increasing reactions to corticosteroids are witnessed every day.

Eosinophil soluble protein levels, eosinophil peroxidase and eosinophil cationic protein in asthmatic patients.

Eosinophil granular proteins are useful eosinophil activation markers in asthmatic patients. In this study, eosinophil peroxidase (EPO) and eosinophil cationic protein (ECP) levels were assessed in different stages of bronchial asthma in 123 patients suffering from intrinsic (n = 42) and extrinsic (n = 81) asthma, with the aim of evaluating the difference in the protein levels between both types of asthma and their importance as a severity marker of the disease. The geometric mean serum level of EPO was 12.3 +/- 2.17 ng/ml (mean +/- SD) in controls, and 38.6 +/- 3.4 ng/ml in the asthmatic patients. Mean ECP levels were 13.22 +/- 1.11 ng/ml in controls and 30.5 +/- 2.38 ng/ml in patients. Depending on the asthma severity, the EPO levels were 30.4 +/- 4.35, 38.7 +/- 5.29, and 54.46 +/- 9.46 ng/ml in mild, moderate and severe asthmatics, respectively, with the differences being significant between the groups of patients with mild and severe asthma (p < 0.001). ECP levels were 24.23 +/- 3.37 in mild, 31.69 +/- 4.21 in moderate, and 37.61 +/- 4.52 ng/ml in severe asthma. There were significant differences in ECP levels between mild and moderate asthma (p < 0.001) and between mild and severe asthma (p < 0.001). Peripheral eosinophil count was 157 +/- 20 eosinophils/mm3 in controls, 334 +/- 35 eosinophils/mm3 in mild asthmatics, 510 +/- 87 eosinophils/mm3 in moderate asthmatics and 658 +/- 72 eosinophil/mm3 in severe asthmatics, with significant differences between all groups (p < 0.05-p < 0.001). Serum EPO and ECP levels and peripheral eosinophil count were significantly greater in patients with active asthma than in patients with silent asthma (p < 0.001). Significant negative correlations (p < 0.001) were found between serum EPO levels and FEV1 (rs = -0.30), MEF25-75 (rs = -0.33), MEF50 (rs = -0.34). There was also a significant (p < 0.001) and negative correlation between ECP levels and FEV1 (rs = -0.31), MEF25-75 (rs = -0.31), MEF50 (rs = -0.32). A good positive correlation was found between peripheral eosinophil count and EPO levels (rs = 0.80, p < 0.001), and ECP levels (rs = 0.67, p < 0.001). We also found a significant positive correlation between clinical score and peripheral eosinophil count (rs = 0.54, p < 0.001), EPO levels (rs = 0.46, p < 0.001) and ECP levels (rs = 0.52, p < 0.001).

Atopic dermatitis and food allergy.

In order to determine the importance of food sensitization in the etiopathogenesis of atopic dermatitis, we performed a study on 74 patients who fulfilled a previously suggested diagnosis criteria. Of these patients, 17.5% presented allergic rhinitis and 62.2% had associated bronchial asthma. We found that in 64.9% of the patients there was a food sensitization, with milk (36.5%), egg (35.1%) and fish (21.6%) being the most frequently involved. We also observed that 34% of the patients were sensitized to Dermatophagoides pteronyssinus and 24.3% to pollen. These sensitizations were confirmed by means of skin tests, specific IgE and antigen-specific histamine release test. The patients underwent a 3-year follow-up in order to find out the clinical evolution once the causal food was avoided and/or a symptomatic treatment was prescribed. The group of patients with no food sensitization was significantly different from the group with food sensitization: in the first group only 20% of the patients presented a very good clinical evolution (asymptomatic), while in the second group, in 71.4% of the patients the symptoms completely stopped. Nevertheless, in the first year follow-up, we found no significant differences between the two groups. In conclusion, a diet avoiding the causal food combined with a suitable symptomatic treatment, led to an important remission of the skin manifestations in children diagnosed with atopic dermatitis.

Effect of immunotherapy on allergen-specific production of sulphidoleukotriene and histamine.

Numerous controlled trials have demonstrated the efficacy of specific immunotherapy, although its mechanism is not completely understood. Few studies have addressed the effects of immunotherapy on the release of mediators. We measured in vitro sulphidoleukotriene (sLT) and histamine release after specific stimulus (Dermatophagoides pteronyssinus or Lollium perenne) in a group of patients under immunotherapy (n = 35) and compared the results with those obtained in a group of allergic patients without immunotherapy (n = 57). SLT quantification was carried out by cellular stimulation allergen test (CAST)-ELISA and we measured the amount of histamine release using a fluorometric method. We found a significant (p < 0.05) reduction of allergen-specific mediator release on the group of patients under immunotherapy treatment. When we studied the group of patients sensitive to D. pteronyssinus we also observed a significant reduction in sLT release after the in vitro stimulus with anti-IgE. In vitro sLT production could be a good marker for follow-up immunotherapy. This study provides more evidence to support the immunological and cellular changes induced by immunotherapy.

Neoallergens in heated soybean hull.

BACKGROUND: During the process of harvest, transport and storage, microbial and mold contamination can raise the temperature of soybeans to 75 degreesC or higher. The purposes of this study were (1) to evaluate the allergenicity of fresh and stored soybean hulls and (2) to ascertain whether heat alters the allergenicity of stored soybean hulls. METHODS: Allergen extracts were prepared from (1) stored soybean hulls, (2) fresh soybean hulls and (3) stored soybean hulls heated to 37 degreesC (E1), 55 degreesC (E2) and 80 degreesC (E3) or kept at room temperature (E4) for 16 h. Individual serum from 68 soybean asthmatic (SA) subjects, 30 nonallergic subjects and two serum pools made from 4 SA sera and 4 sera from asthmatics not sensitive to soybean were studied. All sera and serum pools were assayed for content of specific IgE (radioallergosorbent test) and IgG4 (ELISA). The following additional studies were done for extracts E1-E4: (1) SDS-PAGE, (2) SDS-PAGE/Western blot for specific IgE and IgG4 using both serum pools, and (3) study of the effects of heat on inhibiting activity of the extracts prepared from stored soybean hulls using the pool of SA sera. RESULTS: Test results demonstrated a reduced binding of specific IgE and IgG4 to fresh soybean hull extract compared to stored soybean hull extract, and an increased binding for heated extracts (E1-E3) compared to unheated ones (E4). Moreover, there was an increase in potency for IgE and IgG4 bindings for the heated (E1-E3) compared to unheated (E4) extract, as measured by the amount of protein to produce 50% inhibition. Several protein bands with a molecular weight (MW) higher than 20 kD were absent from the SDS-PAGE for E3 but were present in E1, E2 and E4, and a new protein band (MW 15.3 kD) appeared for E3 only. Two new protein bands, with MWs of 15.3 and 10 kD, which bind specific IgE, were present on Western blot and one of the 3 main soybean hull allergens, probably Gly m 2, disappeared in E3. IgG4 Western blot showed similar results, but only the 10 kD protein band was present. CONCLUSION: The results demonstrate that soybean hull allergenicity is affected by heat, and suggest that the heat generated during storage and transport of soybeans could generate 2 new allergen determinants or increases in epitope exposure as a result of conformational changes. The significance of these new IgE and IgG4 binding proteins has yet to be determined.

In vitro antigen-specific sulphidoleukotriene production in patients allergic to Dermatophagoides pteronyssinus.

BACKGROUND: Sulphidoleukotrienes (slt) are important mediators in allergic diseases that are synthesized after allergen-specific stimulation. OBJECTIVES: The aim of this study is to determine in vitro slt production after allergen-specific (Dermatophagoides pteronyssinus) stimulus of peripheral blood leucocytes and to observe whether histamine release in whole blood with the same allergen correlates with slt production. We also wanted to evaluate whether a correlation exists between the release of slt and histamine and other diagnostic procedures as well as various clinical situations. METHODS: We studied 62 patients sensitive to Dermatophagoides pteronyssinus (Der p), 30 atopic controls and 12 healthy donors. We determined slt production using the CAST-ELISA technique and histamine release using two concentrations of Der p extract (20 and 2 ng/mL). We also carried out quantification of specific and total IgE levels, skin tests and pulmonary function test on each patient. RESULTS: We observed a significantly increased slt release after in vitro stimulation with Der p. There was a significant difference in the slt release between controls and sensitive patients (P < 0.001) and between atopic controls and sensitive patients (P < 0.001). The data are similar to those obtained with histamine release. We noted a positive correlation (P < 0.001) between slt and histamine release (r = 0.71, at 2 ng/mL and r = 0.83 at 20 ng/mL). We also found a positive (P<0.001), although weak (r=0.4 with at 2ng/mL, and r = 0.34 with P = 0.003 at 20 ng/mL) correlation between slt release and specific IgE levels as well as between slt release and skin-test reactivity (r = 0.49 at 2 ng/mL and r = 0.45 at 20 ng/mL; P < 0.001). No significant correlation between slt release and asthma severity was observed, although a trend toward higher slt production in severe and moderate asthma was detected. We found a significant (P<0.001) but weak (r=-0.3) negative correlation between age and slt release. With respect to sex-related differences, we found significant differences (P < 0.05) in slt release between the sexes with a higher slt release in men than in women. CONCLUSION: We conclude that CAST-ELISA for quantification of slt production is a useful in vitro method for diagnosing sensitization to Der p. There also exists a close correlation between slt release and other parameters of allergic sensitization in vitro as well as in vivo.

Study of IgE-dependent sulphidoleukotriene cellular releasability.

Cellular releasability of mediators, as termed by Lichtenstein and Conroy (1), can be triggered by interaction with allergens, anti-IgE antibodies or other agonists. Genetic factors can also influence the cell releasability. We studied 104 subjects, including 92 atopic patients (62 sensitive to D. pteronyssinus and 54 sensitive to Lolium perenne) and 12 healthy controls. Sulphidoleukotriene (sLT) production was measured after allergen and anti-IgE stimulus with CAST-ELISA, and histamine release using a fluorometric method. We found a significant sLT production after anti-IgE stimulation, higher than in basal conditions with medium alone. The sLT production was also significantly higher in sensitive patients than in healthy controls. We found 14.5% of healthy and atopic subjects to be non-responders to anti-IgE stimulus. We also found a positive and significant correlation between sLT production and histamine release. Moreover, we observed a significant positive correlation between IgE-dependent and antigen-specific sLT release. We also noticed a decrease in sLT production and a decrease in histamine release with aging. Male patients had a sLT production significantly higher than female patients. With respect to clinical diagnosis, the group of patients with rhinitis had the highest mediator production. Finally, pollinic patients studied during the spring had a higher sLT production to anti-IgE than those studied out of this season. We conclude that quantification of sLT production after anti-IgE stimulation is a useful method to study cell releasability of mediators and that such releasability is higher in atopic patients than in healthy donors. We must emphasize the usefulness in allergy diagnosis of relying not only on the use of methods demonstrating the existence of sensitization to an allergen, but also of techniques able to quantify the ability to respond to that allergen. In this way we would be able to evaluate the clinical and immunological evolution of patients and to follow up the efficacy of their treatment.

Importance of IgG4 determination in in vitro immunotherapy follow-up of inhalant allergens.

Choosing the right parameters to recommend immunotherapy in allergologic diagnosis is very important. Therefore, other parameters which are independent of the improvement of clinical manifestations and which indicate the evolution of asthma are very useful. Although a decrease in skin reactions was observed in 20% to 25% of patients in previous studies, since the in vitro techniques appeared, their evolution has been observed. Total and antigen-specific IgE evolution, as well as histamine release from basophils, in immunotherapy were followed as we had presented in previous studies. In this work, we studied 151 patients with asthma and rhinosinusitis, 70 of whom were sensitive to Lolium perenne and 81 to Dermatophagoides pteronyssinus. The parameters mentioned above were used, and the patients underwent immunotherapy at three different concentrations depending on the degree of sensitization. The standard concentration was used in the first group; those patients with very high values in radioallergosorbent test (RAST) and histamine release test (HRT) were given the concentration 1:2; and lastly, the largest group of patients, who presented the highest sensitization both in vivo and in vitro, were given the concentration 1:10. As a complement to the mentioned in vitro techniques, the antigen-specific IgG4 determination (blocking antibody) was also evaluated. As we observed in previous studies, skin test, total IgE, antigen-specific IgE and HRTs showed no significant modifications in any of the three groups, in spite of the very positive clinical evolution over the 6 years. Regarding IgG4 follow-up in pollinic patients, a very significant increase was observed, reaching its highest value after 6 years, at standard concentration and with an increase of 49.4%. In conclusion, we think that antigen-specific IgG4 is the only easily available and suitable parameter existing for immunotherapy follow-up.

Sensitization to Alternaria and Cladosporium in asthmatic patients and its in vitro diagnostic confirmation.

In order to determine the prevalence of airborne mould sensitization and the reliability of the in vitro diagnostic techniques in daily practice (antigen-specific IgE and histamine release test), we performed a 3-year study in 2,200 patients diagnosed with rhinosinusitis and/or bronchial asthma. We found mould sensitization in 101 patients, 20% of whom presented monosensitization against airborne moulds, and the rest associated other sensitizations as follows: 53.7% against Dermatophagoides pteronyssinus, 45% against grass pollen and 30% against Olea europea. The most frequently involved moulds in our patients were Alternaria and Cladosporium. Seventy-six percent of the patients presented sensitization against Alternaria, 56% of whom were monosensitized, 26% presented cosensitization to Cladosporium and the remainder were sensitive to more than two moulds. Regarding Cladosporium, the percentage of patients was similar (66%), although only 23% were monosensitized and 46% presented an associated sensitization against Alternaria. We also observed a correlation between skin tests and both in vitro diagnostic techniques, with a relative sensitivity of the specific IgE determination compared to the skin test of 98% against Alternaria and 90.4% against Cladosporium, whereas the relative sensitivity of the histamine release test was 97.4% for Alternaria and 85% for Cladosporium. In conclusion, we think that in order to confirm the etiopathogenesis of the airborne moulds and before an immunotherapy treatment is indicated, the positive skin reactions should be confirmed by means of reliable laboratory diagnostic techniques, such as antigen-specific IgE determination and histamine release test.

Cross-reactivity of Olea europaea with other Oleaceae species in allergic rhinitis and bronchial asthma.

Cross-reactivity between pollen extracts of four species of Oleaceae was studied: olive (Olea europaea), ash (Fraxinus excelsior), privet (Ligustrum vulgare), and lilac (Syringa vulgaris). Thus, 51 patients and 13 atopic controls were studied, by means of intracutaneous skin tests, histamine-release tests against the four extracts, and specific IgE to O. europaea. The proteic content of the four extracts was assessed by SDS-PAGE and immunoblotting, and similarity of all the extracts studied was observed after electrophoresis and immunodetection. Six common bands were found to be responsible for the cross-reactivity, with apparent molecular weights of 49.6, 40, 36.7, 19.7, 16.7, and 14 kDa, respectively. The cross-reactivity was also corroborated by immunoblotting inhibition and FEIA inhibition. The patients had a similar response to the four allergenic extracts used, although the response to Olea was greatest. When the patients were compared by their geographic origin (northern or southern Spain, according to the distribution of areas of olive pollen influence), there were no significant differences between the two groups in skin reactivity, but a higher histamine release was observed for the four extracts in the southern group, although it was significant only for Fraxinus and Ligustrum. This work corroborated the practicality of the diagnostic methods used and the cross-reactivity between the four species studied, as demonstrated by the different methods used. Therefore, we suggest that only O. europaea extract be used in diagnosis and immunotherapy in Oleaceae pollen allergy.

Potentiation of histamine release against inhalant allergens (Dermatophagoides pteronyssinus) with bacterial antigens in bronchial asthma.

In the etiopathogenesis of bronchial asthma, the important role of bacterial infection is more evident every day, favoring inflammation and obstruction, that is, triggering an asthmatic response. We gathered 36 patients diagnosed of bronchial asthma with sensitization to Dermatophagoides pteronyssinus and 32 healthy subjects. Histamine release tests against Staphylococcus aureus extract alone or together with D. pteronyssinus were performed, and the results were contrasted with or without the presence of S. aureus in the nasal secretion culture. We found histamine release against S. aureus higher than 10% only at the highest concentration (200 micrograms/ml) and significantly higher in those patients with positive nasal secretion culture. Regarding histamine release against D. pteronyssinus in the presence of S. aureus, we found a release by coincubation significantly higher than the one obtained from the addition of release against S. aureus and release against D. pteronyssinus, both in patients with negative and with positive nasal culture, at concentrations of 20 and 2 micrograms/ml. In conclusion, we observed a potentiation of histamine release against D. pteronyssinus with S. aureus extract in patients with bronchial asthma. These findings support the important role of the bacterial infection in the etiopathogenesis of bronchial asthma and the importance of a treatment against this infection.

Skin manifestations and immunological parameters in childhood food allergy.

According to Hansen's contact rule, the digestive system should be considered as the main shock organ, yet in food allergy, this is not the case. Very often specific food triggers clinical manifestations not involving the digestive system; that is, reactions are manifested either in the respiratory system, as asthma or rhinitis, or in the skin. In these cases the BALT (broncho-alveolar lymphoid tissue) and GALT (gastrointestinal lymphoid tissue) units play a basic role in the sensitizations. The purpose of this study was to determine the most frequent skin manifestations of food allergy among children, and the most frequently involved foods. We also thought it interesting to evaluate the diagnostic reliability of the different standard immunological parameters utilized by the study team in food allergy. All patients underwent intracutaneous tests with 12 groups of the most frequent food allergens, as well as serum IgE, antigen-specific IgE against foods, and antigen-specific histamine release tests. Antigen-specific IgG4 determination was performed in some cases. The results obtained confirmed previous studies, the most common manifestations being: angioedema (48%), followed by urticaria (31%) and atopic dermatitis (21%). Regarding the frequency of sensitization to different food allergens, in mono- or polisensitization, fish and egg stand out in our environment. Certain food allergens are more frequently responsible for specific skin manifestations. Thus, for fish sensitization, the most frequent skin manifestation is atopic dermatitis (50%); for egg sensitization, angioedema is the most frequent skin manifestation (50%); and for milk, urticaria (50%). Finally, and in agreement with previous works regarding the diagnostic reliability of in vitro techniques, we found that the histamine release test offered the highest percentage of diagnostic reliability. Only for sensitization to milk proteins did antigen-specific IgE demonstrate higher reliability. Once again, we stress that our main problem is the lower reliability of skin tests against food allergens than against inhalant allergens. We emphasize the importance of food as a major factor in the etiopathogenesis of atopic dermatitis, as well as the need to complement the study, when possible, by means of the in vitro techniques described.