Comparison of the Automatic Non-invasive Express Screening Analyser (ANESA)® for Clinical Analytical Parameters

Background: The objective we evaluated the reliability of some haematological and biochemical parameters performed by a non-invasive auto-analyser (ANESA) with those obtained by the standard method of venipuncture (reference method) in patients who went to the clinical analysis laboratory (Municipal-Hospital of Badalona, Spain). Methods: A transversal, comparative and parallel (paired) study was carried out. Two methods of study were practiced for the same subject: a) the reference method of venipuncture (conventional clinical analysis) and b) placement of sensors (comparison method: ANESA device). Consecutive patients older than 18 years, who met certain criteria for inclusion were included in the study during an 8 week period in 2014. The parameters studied were: haemogram (7), glucose, lipids (4), transaminases (2), bilirubin, creatinine and urea. Statistical analysis compared averages for paired groups and reliability of the obtained observations (method: intraclass-correlation coefficient (ICC); individual differences: Bland–Altman method), p<0.05. Results: A total of 195 patients were involved, with an average age of 50.8 years; 65.2% were Original Research Article Sicras-Mainar and Collado-Yurrita; BJMMR, 17(4): 1-17, 2016; Article no.BJMMR.28216 2 women. In paired comparisons, cholesterol (185.4 vs. 179.8; difference: 5.6 mg/dL; p=0.005), cLDL (95.9 vs. 100.5; difference: −4.6 mg/dL; p=0.002) and bilirubin (0.6 vs. 0.5; difference: 0.1 mg/dL; p<0.001) obtained more modest results. Erythrocytes, haemoglobin, haematocrit, platelets, leukocytes, glucose, cHDL, triglycerides, ALT, AST, creatinine and urea reached an ICC>0.90. Lipid parameters (cholesterol: ICC=0.728; cLDL: ICC=0.817) obtained a moderate correlation, whereas lymphocytes (ICC=0.551) and monocytes (ICC=0.546) reached discrete results. Conclusions: Despite of the study limitations, the automatic non-invasive analyser (ANESA) is shown as a reliable and promising screening method in usual clinical practice for most of the analyzed parameters as an alternative to standard blood extraction. However, more studies are required to strengthen the consistency of the results.


INTRODUCTION
Medical diagnosis is a dynamic process that attempts to make ideal decisions in the presence of uncertainty [1][2]. From a functional point of view, a diagnostic test is considered as any procedure done to confirm or discard a presumptive diagnosis in the presence of verisimilitude [2][3]. Nowadays, research into diagnostic tests and their relation to therapeutic innovations are two disciplines which have contributed to greater and faster medical developments [4][5][6][7]. In this way, the advantages of diagnostic methods involve the need for practitioners to have the correct information about their characteristics and applicability in their field of work [8]. The clinical laboratory is the place where analytics are performed, and it contributes to studying, preventing, diagnosing and treating patients' health problems [9,10]. Despite routine use, its performance is not without some impact on patients (intolerance to venipuncture), which can even affect its clinical safety (haematomas, infections, phlebitis, etc.) [11,12]. In this regard, effective and nonaggressive diagnostic methods for patients are considered as ideal in everyday medical practice [13,14].
The functional principle of the Automatic Noninvasive Express Screening Analyser (ANESA) ® is a non-invasive medical device based on thermal measurement (sensors) at certain biologically active points in an organism [15,16]. Information is processed by USPIH software which is the basis for real-time reports of more than 130 parameters of health status. ANESA is based on correlation between heat generation resulting from in vivo chemical reactions of nitrogen, oxygen, hydrogen and carbon and substances without nitrogen [15]. All these processes depend on oxygen supply rate, activity of platelet phospholipids factors, oxygen solubility coefficient, pH and environment temperature. The genetic code manages the cellular elements of blood and biochemical parameters of homeostasis generation [17][18][19].
The evidence available in literature about comparison between parameters resulting from conventional analysis and those obtained by ANESA are very limited. Most of them are in internal documents or informative publications [20]. Assessment of the ANESA device as a reliable method for determination of biological parameters may create great advantages both for patients and professionals, as well as probable resource savings for the National Health System, so the realisation of this study (pioneering in the Spanish State) is relevant. The objective of the study was an evaluation of the reliability of some haematological and biochemical parameters (haemogram, renal function, hepatic function and lipids), which were obtained from ANESA and by the standard venipuncture method in patients who came to the hospital's clinical laboratory.

Study Design and Population
A transversal, comparative and parallel (paired) study was carried out, i.e., each patient was examined by both study methods: a) the reference method of venipuncture (usual clinical analysis) and b) placement of sensors (comparison method: ANESA device). The study population consisted of patients of the Municipal Hospital of Badalona, for whom hospital specialists indicated the need for standard blood analysis. The population, which demands attentive care, is mainly urban and belongs to middle to low socioeconomic section of the population.

Inclusion and Exclusion Criteria
The group of patients was formed consecutively during 8 weeks (February and November, 2014); all patients who met the following characteristics were included in the group: a) age over 18 years old; b) indication by a specialist for routine standard blood analysis (basic health profile or similar), programmed in the Badalona Municipal Hospital clinical analysis laboratory; c) treated in outpatient care, and d) consent to participate in the study voluntarily. Exclusions were: a) displaced subjects or subjects who were outside our responsibility area; b) patients with a preferential or urgent analysis request; c) patients with indication for tests with a specific profile (preoperative, allergies, microbiology, serology, etc.); d) patients in an acute pathological situation (fever or other signs/symptoms) and e) patients with chronic pathology decompensation and/or absence of thermal stabilisation of sensors.

Theoretical Basis of Device
Entropy is the quantitative measure of disorder in a system [21]. The concept comes out of thermodynamics, which deals with the transfer of heat energy within a system [22]. The two principal laws of thermodynamics apply only to closed systems, that is, entities with which there can be no exchange of energy, information, or material. In addition, a method of the ANESA device is based on correlation of heat generation and produced work in a system of internal circulation of the blood [23]. ANESA device is based on Henry's law and Dalton's law, fluid mosaic model [24], model of low-density lipoproteins [25], and totality of knowledge about structure of cell organelles [26][27]. Using all those findings, a biotechnology model of correlation of temperature and lipid exchange was developed. Internal body heat is generated as a result of chemical reaction of nitrogen, oxygen, hydrogen and carbon. The changes of temperatures determine an activation of chemical elements, basically oxygen. Solubility coefficient of oxygen produces alterations on protein and lipid cellular membrane [28][29].
Thermoregulation is sophisticated regulatory process of energy metabolism and hemodynamics, originating from interaction of hippocampus, hypothalamus and pineal gland, basing on energy reactions of adenosin tris fosfato and transmembrane choline-containing phospholipids [30][31][32][33]. In this way, thermoregulatory reactions such as sweating and shivering enable the body to keep its core temperature constant. This process depends on the rate of oxygen supply, the activity of phospholipid factor of thrombocytes, solubility coefficient of oxygen, pH medium and temperature. Thermal component of those reactions depends on cholinergic and adrenergic neurotransmitter systems, related to carotid body and formation of charge-transporting interrelation at the level of thyroxin and imidazole protein receptor of a red blood cell and peroxisome proliferator-activated (PPAR) [34][35][36]. PPAR together with red blood cells takes part in formation of pH of artery blood, according to temperature parameters of interaction of opioid receptors (α, β, γ) in cholinergic and adrenergic regulatory mechanisms of endogenous alcohol synthesis, resulting from the interaction of metabolic processes of cholesterol, vitamin B1 and D, glucose, lactic acid, ubiquitin, intestinal synthetase and α1trypsin. Temperature and pH of arterial blood are connected with lymphoid myeloid complex, which is presented in all organs and hematopoietic system [35][36].
In summary, metabolic response on the reactions in organism corresponds to the ratio of the sum of temperatures in the carotid bifurcation (carotid body) to a temperature parameter in abdominal area. In this case it is proved that the temperature is the final stage during biochemical and biophysical changes on the level of lysosomes, cytosol and mitochondria. It depends on metabolism of amino acids, phospholipids and cholesterol, and water metabolism. Changes in temperature parameters of mentioned areas are caused by changes in blood circulation and depend on blood circulation in organs, which are controlled by hypothalamus and tyrosine kinases. The method in whole is widely described in monograph "Thermoregulation of an organism and vegetovascular paroxysms" [37]. The certificates, patents, allowable limits of performance and quality controls of the device are described in the literature reviewed [38][39]].

Study Groups and Predetermination of Sample Size
A single group of patients was examined by both methods (at first using ANESA and then by conventional blood sampling) to obtain haematological and biochemical parameters (haemogram, renal function, hepatic function and lipid profile). The calculation of the sample size was realised as a function of the variability of the average of parameters at 10%, assuming a random error of 5% and an estimated accuracy of 1.5%. The minimum number of patients necessary to make the comparison between both methods was 170. The statistical power for the model was higher than 80%.

Organisational Procedure -Personnel Training
A multidisciplinary working group was established of nine professionals (two nurses, two intern doctors and a family doctor, one laboratory supervisor, three heads of service and one coordinator) for planning the study. A technical training course was conducted for these professionals, in order to learn the physiological, functional and organisational fundamentals of ANESA. Selection of subjects was performed from a basic list (see inclusion/exclusion criteria) of patients scheduled for usual blood analysis. Note that previously the whole hospital had been informed (via corporate intranet) about realisation of the tests especially (personalised information in a group) reception professionals, administrative personnel in the outpatient department, laboratory staff and personnel in the day hospital. Patients who were accepted to participate in the study were informed about the testing procedure by telephone (a week before); they were invited for the planned day of testing (reduction of waiting time) to the hospital (third floor waiting room) and were called out to the testing room every 15 minutes; altogether 8-10 patients per day were tested.

Preparation of the Patients and Detail of the Procedure
On the day of testing, healthcare staff invited a patient and accompanied him or her to the testing room in the day hospital. There, the patient was informed about the testing procedure again, inclusion and exclusion criteria were checked once more and an informed consent form was offered to the patient for signing. During the test, a doctor and a nurse were always in the room. The patient was placed in a reclining armchair (supine) and was familiarised with the sensors (direct contact) to decrease emotional state. Five sensors were placed on biologically active points: a) bifurcation of the carotid artery left and right (two points); b) axillary region left and right (two points) and c) umbilical region (Fig. 1). Heart rate, age and weight had been determined previously and entered into a computer using a keyboard, along with the gender of the patient. After that, measurement by the ANESA device software was started; after finishing that, blood was sampled.
The estimated time of measurement for each test was 4-9 minutes depending on stabilisation of temperatures for real-time result readings. Those cases in which temperature values were not stabilised adequately were not considered as valid data for inclusion in the analysis and such patients were excluded. The five sensors connected to the analyser measure temperature from the reference points of a patient with an accuracy of not less than 0.5°C. The sensors send temperature parameters to the ANESA central processing unit. Calculation of blood parameter data is done with a special examination algorithm named as the Malykhin-Pulavskyi method (Ukrainian patent No. 3546 A61B5). During examination, there exists no harmful influence from the analyser to patients (non-invasive method). As the analyser determines the influence of environment on a patient's health status, a test is not recommended if: a) the environment temperature is higher than 27°C or lower than 20°C; b) relative humidity is higher than 80% at 25°C; c) sunshine and/or air conditioning flow are directed at a patient or d) strong electrical or magnetic fields exist in the environment. The device's enclosure was cleaned with a soft welldrained soapy cloth. Disinfection of sensors was done with alcohol wipes (96% alcohol

Information Confidentiality
Confidentiality of records (anonymous and differentiated) was respected according to the Data Protection Organic Law (15/1999 Law from December 13). All patients were informed about the nature of the study (patient information sheet). They were also informed that all data obtained by ANESA apparatus would be valid only for the study, i.e., only information for estimation of health status and possible medical acts, obtained by standard blood analysismight be taken into account. All participants signed the informed consent form. The study was approved by the Clinical Investigation Ethics Committee of Germans Trías and Pujol University Hospital in Badalona, Spain.

Statistical Analysis
A descriptive analysis of all evaluation parameters was performed separately, using absolute and relative frequency tables in the case of qualitative variables and statistical averages, standard deviation, Pearson linear correlation, percentiles and/or confidence intervals (IC 95%) in the case of continuous quantitative variables. Distribution normality was verified through a Kolmogorov-Smirnov test. For parameter comparison of both methods (ANESA vs. standard laboratory analysis), a bivariate analysis was done through average comparison for paired groups (T-Student). Internal consistence was analysed by Cronbach's alpha coefficient (based on the average correlation of the items), reliability of the clinical observations through intraclass correlation coefficient (ICC) and analysis of individual differences through Bland-Altman graphs [40]. In the study, an acceptable comparison for ICC values > 90% (level of medical decision) was considered. SPSSWIN version 17 software was used, establishing a statistical significance for values of p<0.05.

RESULTS
The total number of patients involved was 195. Of those, N=2 (1.0%) refused to participate in the study, N=13 (6.7%) did not meet the initial inclusion criteria, and in N=10 (5.1%) temperature stabilisation was not reached (thermal regulation), which was required to perform the non-invasive test; finally, 172 subjects were analysed. The baseline characteristics of the patients are detailed in Table 1. Average age was 50.8 years old and 65.2% were women.  Finally, analysis of the reliability and internal consistency of observations between both measuring methods studied (ANESA device vs. standard laboratory analysis), is described in Table 3. In the 17 studied parameters and comparing both study methods (ANESA device vs. standard laboratory analysis), the relationship between internal consistency and reliability was equivalent. Erythrocytes, haemoglobin, haematocrit, platelets, leukocytes, glucose, cHDL, triglycerides, ALT, AST, creatinine and urea reached an ICC>0.90. Lipid parameters (cholesterol: ICC=0.728; cLDL: ICC=0.817) obtained a more modest correlation; meanwhile lymphocytes (ICC=0.551) and monocytes (ICC=0.546) did not reach expected results. Analysis of the individual differences though Bland-Altman graphs for each of the analysed parameters is shown in Fig. 2. The statistical sub-analysis performed by age and gender average, reliability and consistency of the observations remained without statistically significant differences (similar results).

DISCUSSION
Results showed that the non-invasive ANESA device is a reliable and advantageous method for usual clinical practice in comparison with standard laboratory analysis parameters, being an alternative to the usual blood extraction screening method. It should be noted that few published studies exist which compare these two methods for obtaining parameters in real conditions (which makes the comparison of results difficult), which is why the present study must be interpreted as strong, since it offers relevant information for an alternative to the usual laboratory analysis. However, without an adequate standardisation of methodology for measurement of variables, the results must be interpreted wisely, causing us to be cautious in the validity of the results in general practice.
The device could not substitute the usual clinical analysis laboratory, but may be used as a complementary method for patients with noncomplex clinical pathologies (it measures 130 parameters) in primary care centres, special medical institutions, preventive care departments and centres for screening and monitoring of chronic diseases with low complexity (such as dyslipidaemia, diabetes, etc.). In addition, the ANESA device could be very useful in urgent or complex situations as it combines parameters which allow estimation of gas analysis, spirometry or even other analysis as related to cardiac, hepatic, pulmonary or renal disorders. They have not been the objectives of this study, but that is a promising way forward for the future; it offers alternatives that will be analysed and verified.   The statement above could be comparable in the context of the obtained results from the study. In our case, in people with noncomplex clinical pathologies, erythrocytes, haemoglobin, haematocrit, platelets, leukocytes, glucose, cHDL, triglycerides, ALT, AST, creatinine and urea reached optimal results, whereas lymphocytes and monocytes did not reach expected results. The last may be caused by a lack of conversion in the ANESA's algorithms or by random effects. A special mention must be made about lipid parameters, which obtained a modest correlation. According to the opinion of scientific associations, lipid parameters have a high variability margin, so their interpretation must be performed taking into account potential modifying factors. So, factors affecting ANESA's algorithm as well as the results should be improved in order to reduce the variability margin compared with standard laboratory analysis [44-45].

Study Limitations
Possible limitations of the study are disease complexity, possible bias of classification of patients (inter-individual variability) and effective measurement of variables (in vivo vs. in vitro). Moreover, some bias may occur because of: a) an imperfect reference test, and as there is no good reference test, the one available test is used (although this test does not classify well and does not always differentiate healthy and sick correctly), and all reference tests are subject to a variability margin (technical, inter-individual, etc.) and b) differences in result interpretation.
This study was realized with patients attending outpatient care (scarce clinical complexity), so this situation should be considered as a limitation of the study. Lipids results were slightly discordant. This fact may be due to several factors: a) a random phenomenon, b) a lack of reliability in the method, c) the own biological variability of the biochemical parameters analyzed in the laboratory [46], and / or other unmeasured variables in the study. However, the most important limitation is a lack of external validity of the study, so the generalisation of results must be interpreted cautiously. In our study, the ICC was used to compare quantitative data. Although it is a valid method for analysis; It could have also used the regression model Passing-Bablok (homogeneity of variances is required) [47]. Another study limitation is the lack of comparison with literature due to there is no published recent studies, for the moment.

Future Directions
In future researches we should answer these inaccuracies. It is an investigation field with a high potential in clinical practice. The device's merits, such as use of a non-invasive procedure, with results in real time and at very low cost, are only some of the advantages of this type of device.

CONCLUSION
Despite of the study limitations, the automatic non-invasive analyser (ANESA) is shown as a reliable and promising screening method in usual clinical practice for most of the analyzed parameters as an alternative to standard blood extraction. However, more studies are required to strengthen the consistency of the results.