Detection of angiospastic disorders in the microcirculatory bed using laser diagnostics technologies

Irina N. Makovik*††, Andrey V. Dunaev*, Victor V. Dremin*, Alexander I. Krupatkin, Viktor V. Sidorov, Lyudmila S. Khakhicheva, Vadim F. Muradyan, Olga V. Pilipenko*, Ilya E. Rafailov¶ and Karina S. Litvinova||** *Biomedical Photonics Instrumentation Group Scienti ̄c-Educational Centre of \Biomedical Engineering" Orel State University named after I. S. Turgenev Orel 302026, Russian Federation Priorov Central Research Institute of Traumatology and Orthopaedics Moscow 127299, Russian Federation SPE \LAZMA" Ltd, Moscow 125252, Russian Federation Orel Regional Clinical Hospital Orel 302028, Russian Federation ¶School of Engineering and Applied Sciences Aston Institute of Photonic Technologies Aston University, Birmingham, B4 7ET, UK ||Optoelectronics and Biomedical Photonics Group Aston Institute of Photonic Technologies Aston University, Birmingham, B4 7ET, UK


Introduction
The evaluation of the microcirculatory bed functional state in the upper limbs presents itself as a major problem in modern medical diagnostics.Its essence lies in the development and improvement of diagnostic methods, which would contribute to a better de¯nition of reserve and adaptive capabilities of the microcirculatory bed, as well as the timely detection of various pathologies.
Angiospastic disorders are one such pathology.These disorders provoke vasospasm of vessels and represent a reversible localized or di®use vasoconstriction of arteries or smaller blood vessels. 1 Today, there are a distinguished number of diseases of the angiospastic nature, such as Raynaud's disease, acrocyanosis, and livedo reticularis. 2hese disorders may be in the form of angiospastic attack with the occurrence of reversible ischemia, without compromising the integrity of the vascular system and tissues.4][5] Thus, the expression of data processes will increase with an increase of the interval between tissue oxygen demand and the level of blood °ow.
Angiospasm plays an important role in the pathogenesis of a variety of diseases.It can appear as an independent disease, 6 appear under the in-°uence of external factors, various chemicals and medicinal drugs, 7 and can develop in the background of such diseases as scleroderma, 8,9 systemic lupus erythematosus 10,11 and rheumatoid arthritis. 12,13Involvement of the vessels into the pathological process, in the background of existing connective tissue system disorganizations can lead to even greater aggravation of the disease course and outcome and the development of tissue hypoxia.This, in most cases, will in°uence the approach to treatment and prognosis of the disease as a whole.
Thus, a timely detection of angiospastic microcirculatory bed disorders as well as tissue oxygen supply and consumption changes (transcapillary exchange) is important, both in the manifestation of their primary characteristics (when pathological changes are still reversible) and in various pathologies, in order to prevent aggravation of diseases and reach better treatment.
Certain optical noninvasive technologies, namely the methods of laser Doppler °owmetry (LDF), 14,15 tissue re°ectance oximetry (TRO) 16 and pulse oximetry (PO), 17 appear to have potential for the study of the microcirculatory bed functional state and the detection of angiospastic disorders.These methods allow for in vivo evaluation of the peripheral circulation level, transport dynamics, the value of blood oxygen saturation in the microvessels and the percentage of oxyhaemoglobin in arterial blood.
Furthermore, the above methods are capable of providing vast amounts of physiological data.This data comes in the form of frequency ranges (0.0095-1.6 Hz), which re°ect the di®erent mechanisms of regulation.There are currently ¯ve categorized frequency ranges. 189][20] Active mechanisms of regulation are myogenic (0.047-0.145Hz), transmitting the activity of vascular smooth muscle cells, [21][22][23] neurogenic (0.021-0.046][27] Thus, with the use of this mathematical apparatus, it possible to evaluate the main factors provoking vasoconstriction and angiospastic disorder.Analysis of endothelial °uctuations reveals the presence of disturbances in the vascular endothelial function. 28As is known, these violations are manifested in the form of a decrease in the concentration of endogenous vasodilators acting on the endothelium, [29][30][31][32] namely, in reduced synthesis of NO, 33,34 and also in an increase of vasoconstrictor substance activation, such as endothelin-1. 35,36According to the analysis of oscillation mechanisms of neurogenic and myogenic genesis, it is possible to judge the presence of other factors, which induce angiospastic disorders, such us activation of the alpha-adrenoceptor, 37 increases in smooth muscle contractility and about launching of di®erent vasomotor re°exes in the result of activation of the sympathetic nervous system. 6,38n this regard, evaluation of the prospective combined use of these methods was proposed, in order to analyze all the changes arising from angiospasm; beginning with blood °ow violations and ending with the development of hypoxia. The combined application of the suggested diagnostic methods and various functional tests contributes to the increased overall level of informative diagnostic data.Their use contributes to the evaluation of not only the general condition of the microcirculatory bed, but its reserve and adaptive capacities.One provocative test, used in the study of the human body's functional state, is the cold pressor test (CPT).This test is carried out through complete immersion of the upper or lower limbs of the body in a container with cold water.The water temperature during the experiments may be from 0 C to 15 C, with study durations reaching up to 30 min. 39The stimulus, which in the case of the CPT immersing in cold water, provokes many interrelated processes.Namely, these are the changes in blood pressure and heart rate, [40][41][42] irritation of thermoreceptors, activation of the sympathetic nervous system, 43,44 constriction of muscle containing vessels, 45 synthesis stimulation of various neurotransmitters (such as noradrenaline, dopamine 46 ), etc.After the termination of cold exposure in a normal functional state of the body and the absence of disorders, there is a stabilization and recovery of all body processes.Presence of these disorders exhibits no such recovery.
][49] Thereby, the aim of this study was to evaluate the possibility of combining the LDF, TRO and PO methods with the cold pressor test for the analysis of reserve and adaptive abilities of the microcirculatory bed in the upper limbs and detecting the presence of angiospastic disorders.

Inclusion and exclusion criteria
To evaluate the combined use functionality of the LDF, tissue re°ectance oximetry, pulse oximetry and cold pressor test methods, experimental studies were conducted on 32 healthy volunteers and the Orel Regional Clinical Hospital (Orel, Russia).The study was approved by the local Committee for Human Biomedical Research Ethics and was carried out in accordance with the principles outlined in the 2002 Declaration of Helsinki by the World Medical Association.The study protocol and its purpose were explained in detail to each subject and the informed consent was obtained from all of the subjects.
The group of healthy volunteers included 16 men (average age -22AE1 year) and 16 women (mean age -22AE2 years).The participating volunteer group was collected through an internal university advertisement circulated by physical posters and noti¯cation email.According to the preliminary interview, these people did not exhibit disorders of the cardiovascular system and led healthy lifestyles.
Selection of patient volunteers was assisted in by the attending physician, based on the medical history, analysis of the disease and any co-morbidities, results of prior studies conducted using di®erent diagnostics methods and in the presence of laboratory blood test changes (increases in laboratory parameters such as rheumatoid factor, antinuclear factors, thrombocytosis and anaemia).Additionally, patient complaints about pain in their ¯ngers, increased sensitivity to cold and cold induced discoloration were also taken into account.
The main characteristics of the patient group are presented in Table 1.The data-range averages, as well as the minimum and maximum values, are provided for each parameter.
As described previously, disorders of upper limb microcirculatory bed are most commonly found as one of the forms of rheumatological pro¯le disease pathologies.These diseases are more common in the elderly.It is thus necessary to clearly di®erentiate between a healthy state and one with microcirculatory bed disorders.A group of healthy young volunteers was recruited as a control to ensure an \extreme" state of good health, as they would present the lowest chance of exhibiting any undesired physiological conditions.

Study protocol
The laser analyzer of blood microcirculation \LAKK-OP" was used to register parameters of the microcirculatory bed on healthy volunteers.The multifunctional laser diagnostic complex \LAKK-M" (SPE \LAZMA" Ltd, Russia) was similarly employed on the patients.These diagnostic devices utilized identical measurement channels and are designed to study the state of peripheral blood °ow.This is achieved by a simultaneous radiation of sample tissue through super¯cial contact with optical sensors.A 1064 nm laser wavelength for LDF and 532 nm and 635 nm radiation wavelengths for the TRO and pulse oximetry methods were delivered and received by the optical sensors.The location of these sensors on the ¯ngers during the experimental studies on volunteers using the \LAKK-OP" is presented in Fig. 1(a); the experimental setup for the study on patients using the \LAKK-M" is presented in Fig. 1(b).
The LDF 3.0.2.384 and LDF 3.1.1.407software packages were used for the frequency analysis of the passive and active microcirculatory regulation mechanisms.These software packages use a continuous wavelet transform, with the Morle complex valued wavelet being used as the analyzing wavelet. 50,51hree basic (background) tests (BT), 5 min duration each, were involved in the experiment using the CPT: BT1 was recorded before CPT, BT2 immediately after and BT3 20 min after CPT completion.The temperature of water during the CPT was 14.8AE0.2C. A contactless electronic thermometer \Sensitec NB401" was used for the temperature control of water and volunteer skin.The LAKK device measurements were performed on the skin pad (palmar surface) of the right middle ¯nger.This area was chosen because it is rich in arteriolarvenular anastomoses (AVA), which depend on the sympathetic innervation as well as autonomic and sensory nerve ¯bers.Analyzes of the changes of parameters in this area allow to diagnose various microvascular disorders, which begin at the level of the microcirculatory bed of the ¯ngers, including angiospastic disorders.Moreover, the area with AVA has lower spatial variability due to a low disparity in skin capillary density, which ensures lower variability of parameters and better reproducibility of data. 52,53ll measurements were performed in conditions of physical and mental rest 2 h after a meal.
Volunteers also underwent a preliminary adaptation to room temperature 24-25 C for 15-20 min in a sitting position, with the right arm on the table at heart level.The adaptation of volunteers to standard room temperature and abidance of study protocol during all measurements reduces the in-°uence of di®erent factors on results of diagnosis.

Measured and calculated parameters
Basic microcirculatory bed parameters were registered during experimental studies, providing a vast array of information.Due to the expediency of the complex diagnostic of the upper limb microcirculatory bed, the oxygen extraction (OE) and the rate of oxygen consumption (OC) were calculated based on the measured parameters obtained by a methodology relying on the analysis of LDF and TRO determined amplitude-frequency spectra. 54,55The calculation of these parameters makes it possible to evaluate the changes in the tissue oxygen supply and consumption.In this case, the value of the OE represents the process of oxygen di®usion from the arterial blood into the tissue.The calculation of the rate of OC gives an indication of the dynamics of oxygen utilization by tissues.
where S v O 2 is venous blood oxygen saturation.
To determine the values of venous blood oxygen saturation, the amplitude of oscillations associated with passive mechanisms of S t O 2 regulation were analyzed, namely: the amplitudes of the cardiac A (S t O 2 ) c and respiratory A(S t O 2 ) r oscillations.
If the condition is met, then the venous blood oxygen saturation is calculated as follows: where A(S t O 2 ) c , A(S t O 2 ) r are the maximum oscillation amplitudes of cardiac and respiratory origin of the tissue oxygen saturation.This variant is predominant in most cases recorded on skin with AVA's.
In the case of the resonance of tissue oxygen saturation oscillations, when high-amplitude oscillations are observed only in one of the active regulatory mechanisms against a sharp suppression of oscillations and a decrease of amplitude in the other ranges, the calculation of S v O 2 has several features.In the case of the oscillation resonance in the general myogenic or respiratory ranges, the venous blood oxygen saturation is calculated as follows: where BI(S t O 2 ) is bypass index, is calculated using the S t O 2 -gram: are the amplitudes of the oscillations of the tissue oxygen saturation in neurogenic and myogenic ranges.The rate of OC is calculated based on the analysis of the LDF-gram oscillations and the previously calculated value of venous blood oxygen saturation: where I mnutr is the proportion of nutritive blood °ow in the general microcirculation (nutritive blood °ow).This value characterizes the blood °ow in the capillaries and is calculated by the formula: where BI(I m Þ is the bypass index, characterizing blood °ow in the AVA.This parameter is calculated using the LDF-gram and the formula: The component BI(I m Þ 1 is calculated by formula (5) using perfusion data (I m Þ.In the case of dominance of blood °ow oscillations, associated with endothelial regulation mechanisms in the calculation, the oscillation amplitude of neurogenic origin (A n Þ is replaced by the amplitude of oscillation associated with endothelial mechanisms (A e Þ during calculation of the bypass index.BI(I m Þ 2 is calculated by the formula: where AðI m Þ pass is the maximum oscillation amplitude of blood °ow associated with passive mechanisms of regulation (oscillation of cardiac or respiratory origin).BI(I m Þ 2 is taken into account in the calculation of bypass index if BI(I m Þ 2 ! 1. 54 Thus, the calculation of these parameters makes it possible to assess the perfusion on nutritive (capillaries) and shunt (AVA)-based blood °ow pathways of the microcirculatory bed.
To estimate the oscillatory component of microvascular tone, the values of endothelial (ET), neurogenic (NT) and myogenic (MT) tone were determined.
where is mean square deviation of the blood microcirculation index (I m Þ; AðI m Þ ethe greatest value of oscillation amplitude of perfusion in the endothelial range.
where AðI m Þ n is the greatest value of oscillation amplitude of perfusion in the neurogenic range.
where AðI m Þ m is the greatest value of oscillation amplitude of perfusion in the myogenic range.
In addition to analyze the oscillatory component of microvascular tone, the calculated endothelial and neurogenic tones contribute to the evaluation of alpha-adrenoreceptor activity and the synthesis of endothelial factors.The calculation of MT enables the analysis of the degree of precapillary sphincter closure, which are under the control of the internal myogenic regulatory mechanisms 56,57 and play the main role in the regulation of the exchange surface area and the number of functioning capillaries.
Thus, the use of this method enables a more detailed analysis of the microcirculatory bed's functional state and its structural and functional units, while also contributing to the evaluation of di®erent processes in microvessels; from analysis of the blood transport function to changes in the dynamics of oxygen utilization by tissues and the identi¯cation of factors that have the greatest impact on these processes.

Statistical analysis
All data obtained from the results of the research were checked for normal distribution using the Kolmogorov-Smirnov test and homogeneity of variance using the Levene's test.Analysis of the statistical signi¯cance di®erences of these data was performed using a two-factor ANOVA with repetition (Two-Way Repeated Measures ANOVA).The probability was statistically signi¯cant with a p-value of less than 0.05.The choice of this statistical analysis has been associated with the fact that the experimental study has a two-way design (the ¯rst factor is phase of the cold pressor test, i.e., BT1, BT2, BT3, and the second factor is participant type, i.e., healthy volunteers or rheumatic patients).The use of this test reduces risks of false positives due to multiple testing, which could occur with repeated use of other statistical methods of data processing.

Results and Discussion
The cold pressor test causes signi¯cant changes in microcirculatory-tissue system parameters of the upper limbs.Figure 2 shows examples of registered LDF-and TRO-graphs and their amplitudefrequency spectrums before (Figs.2(a) and 2(c)) and after (Figs. 2(b) and 2(d)) the cold pressor test.

Statistical analysis of groups
A detailed statistical analysis of the parameter changes for each individual in the two groups displayed a di®erent response of the microcirculatory bed parameters upon the cold exposure, with a di®erent recovery of parameters after CPT.A partial or complete recovery was observed, with individual cases of no recovery of the parameters, which could indicate both the normal state of the microcirculatory bed and the presence of angiospastic disorders.It should be noted that this was a contrast to the statistical analysis of data obtained for the groups of 32 healthy volunteers and 33 patients with diseases of a rheumatological pro¯le.Not individually assessing the volunteers seemed to conceal any signi¯cant di®erences in each basic test.

Additional analysis of measured and calculated parameters
To separate the groups by the criterion of the presence or absence of angiospastic disorders, an additional analysis of the measured and calculated parameters was carried out.The results of analysis showed that for the identity of the possible angoispactic disorders of the microcirculatory bed and related abnormalities, it is necessary to assess changes of the I m , MT and OC after CPT.These parameters were selected because the shift of the balance between vasoconstriction and vasodilation, which occurs under the in°uence of many factors, leads to a change of functioning of the basic structural and functional units of the microcirculatory bed and decreases blood °ow in larger vessels (arteries, arterioles) and their capillary components.
The evaluation of the blood microcirculation index allows conclusions to be made regarding the total decrease of blood °ow during these disorders.At the same time, the analysis of changes occurring in the MT and rate of oxygen consumption will give information about the degree of reduction in the number of functioning capillaries, the area of exchange and the decrease in supply and consumption of oxygen to tissues.
The analysis of published data shows that time for stabilization of the all regulatory mechanisms after the cold pressor test can range from 15 min to 30 min depending on the temperature of the in°uencing factor and cooling time. 39][60][61] The threshold value by which to diagnose angiospastic disorders in this case was from 40% to 60% of the initial level. 61

Proposed criteria for identify of angiospastic disorders
The presence or absence of angiospastic disorders and the possible tendency to angiospasm were suggested based on these new criteria for separation of the diagnostic results.These criteria take into account not only the change of parameters, but also increase (decrease) of the analyzed parameters during CPT in relation to their initial level: where I m1 is initial value of blood microcirculation index; I m2 is increment of blood microcirculation index after the CPT; I m3 is ¯nal value of blood microcirculation index.
where MT 1 is initial value of myogenic tone; MT 2 is value of myogenic tone after the CPT; MT 3 is ¯nal value of myogenic tone.
where OC 1 is initial value of rate of oxygen consumption; OC 3 is ¯nal value of rate of oxygen consumption.
If the ratio of the ¯nal and initial increment I m values relative to the value immediately after the CPT is more than 50 %, i.e., and ratio of the ¯nal and initial increment MT values relative to the value immediately after the CPT is more than 50%, i.e., and if OC reaches the initial level, then a normal MTS state of limbs/extremities is diagnosed.
If the ¯rst two criteria are not met, tendency towards angiospasm is diagnosed.If at least one of the ¯rst two criteria is not satis¯ed, repeat the diagnostic procedure.
To identify the possible reasons for the tendency towards angiospasm, it is necessary to evaluate the amplitude ratios of the cardiac and respiratory blood °ow oscillations 20 : where AðI m Þ c , AðI m Þ r are the maximum oscillation amplitudes of cardiac and respiratory origin of the blood microcirculation index.If 20 min after the CPT, when the ¯rst two criteria are not satis¯ed and the value of the amplitude ratio of the cardiac and respiratory blood °ow oscillations is greater than 1, then we associated the cause of the possible tendency towards angiospasm with increased myogenic tone.If the amplitude ratio value of the cardiac and respiratory oscillations of blood °ow is less than or equal to 1, then we associated the possible cause of angiospasm, in addition to the enhanced MT, with venous stasis.The proposed criteria of the normal state and the tendency to angiospasm, as well as the possible causes of disorders, are presented in Table 2.
It should be noted that the stagnant causes of disorders are almost always combined with high MT.Their detection is a manifestation of the am-pli¯cation of the myogenic activation, which leads to more signi¯cant haemodynamic disorders in the form of venous stagnation.The key distinctive mechanism leading to angiospasm is the myogenic component.

Analysis of groups taking into account the proposed criteria
Using as a criterion for the selection of healthy volunteers without possible microcirculatory bed disorders, the above-described criterion of microcirculatory bed functional state recovery of at least 50% of the level before the CPT, the following results were obtained The group of 32 healthy volunteers exhibited two typical responses: in 24 volunteers by the end of the study there was complete parameter recovery, in ¯ve volunteers this recovery did not occur.Also a small group of volunteers (n ¼ 3) was identi¯ed, in which no signi¯cant changes in the microcirculatory Table 2.The proposed criteria of the normal state and the tendency to angispasm, as well as the possible causes of disorders.

No. Criteria
The normal state Myogenic reasons Myogenic-stagnation reasons bed parameters occurred throughout the study.As described in this work, 62 this response is one of the possible responses to cold exposure.The data for these volunteers was also included in the group of volunteers without angiospastic disorders.Among the 33 patients studied according to the proposed criteria, 18 patients were diagnosed to have a relatively normal functional state of the microcirculatory bed, 15 patients were diagnosed to have a possible tendency to angiospasm.Further, the statistical analysis of the data between the ¯rst and second groups in each sample showed signi¯cant di®erences in parameters.

Statistical analysis of relatively normal state and state with angiospastic disorders
To highlight the di®erences between relatively normal state and presence of angiospastic disorders, detected using the proposed approach, a comparison was conducted.For this a statistical analysis of experimental data was carried out for healthy volunteers without identi¯ed angiospastic disorders (relatively normal state), and patients with identi-¯ed angiospastic disorders.Figure 3 shows the results of statistical analysis of data from healthy volunteers without identi¯ed angiospastic disorders (relatively normal state) (No. 2) and patients with identi¯ed angiospastic disorders (No. 2): the index of blood microcirculation (Fig. 3(a)), tissue oxygen saturation (Fig. 3(b)), MT (Fig. 3(c)) and the rate of oxygen consumption (Fig. 3(d)).The result of statistical analysis for all measured and calculation parameters are presented in Table 1, located in the Supplementary material of this paper.
As can be seen from the data after a stimulus in the form of the cold pressor tests, there is a decrease in peripheral blood °ow in both groups.The change of the blood °ow is due to the development of vascular smooth muscle cell constriction in the vessel wall (arteries and arterioles) as the result of activation of the sympathetic nervous system and alpha-adrenergic receptors. 37,63Several studies have indicated the possible participation of endothelial cell vasoconstrictor substances such as endothelin-1 35,64 in this reaction.Against the background of these processes, increases in MT and decreases in nutritive blood °ow were observed in both groups.
Analyzing the changes in these parameters, it is evident that after cold exposure in both the functional states of the microcirculatory bed (with angiospastic disorders and in a relatively normal function state), there is an increase in spasms of precapillary sphincters.As a result of this process, the number of functioning capillaries decreases, which ultimately a®ects the reduction in capillary blood °ow.These processes cause a reduction in tissue oxygen utilization.
It should also be noted that, despite the higher initial level of total blood °ow in patients with angiospastic disorders in relation to healthy volunteers without angiospastic disorders, the analysis of shunt blood °ow values and the nutritive blood °ow between these groups shows signi¯cant di®erences.In healthy volunteers, the greater contribution to the total blood °ow is introduced into their capillaries.Patients did not have such a signi¯cant di®erence in the values of these parameters.Additionally, the patients have a lower value in the rate of oxygen consumption.That, in combination with the previously described processes, may indicate initially reduced level in oxygen utilization by tissues.Thus, comparing the parameters between two groups, even conditions of relative rest without provocative impact make the detection of changes in these parameters possible, which may indicate the possible presence of a disorder.
After the termination of cold exposure, the recovery of parameters in the group exhibiting a relatively normal state of microcirculatory bed (without a tendency to angiospasm) and in the group with a tendency to angiospasm occurs in di®erent ways.
In the group with a relatively normal state, after cold in°uence there is an increase in local temperature and dilatation (widening) of blood vessels, these processes constituting the so-called cold vasodilation reaction.As a result of this reaction, blood vessels lose the ability to shrink, dilate and become a passive vascular bed. 65This process may be associated with increased synthesis of endothelium vasodilating substances such as nitric oxide (NO) and other mediators of vasodilation.20 min after the CPT there is a stabilization of all oscillatory blood °ow mechanisms, the decrease of MT and the activation of perfusion.By reducing spasms of precapillary sphincters, the number of functioning capillaries increases and processes of transcapillary oxygen exchange are restored.This is con¯rmed by the fact that the nutritive blood °ow and the rate of oxygen consumption increase.
In the group with a tendency to angiospasm, recovery of microcirculatory bed parameters did not occur.This process may be the result of a shifting balance between vasoconstriction and vasodilatation towards the former, due to intensive and prolonged spasms of the vascular wall and reduced concentration of endogenous vasodilators acting on the endothelium, namely reducing the synthesis of the NO.That may be associated with endothelial dysfunction. 33As a result, the value of the total perfusion decrease and MT increases.All these processes lead to an even greater reduction in the number of functioning capillaries, resulting in a decreased proportion of nutritive blood °ow and rate of oxygen consumption.Analysis of the possible reasons of angiospastic disorders showed that in most cases they were related to increased MT, with only two patients displaying venous stasis on the background of increased MT.
The additional information about values of analyzed parameters is presented in Table 1 located in the Supplementary material of this paper.

Relationship of angiospastic disorders state of patients with the diagnosis and the results of laboratory studies
The analysis of the data and their relationship with the diagnosis and the results of laboratory studies have shown that there are several factors, which potentially indicate and con¯rm the presence of the possible angiospastic disorders in these patients.Four of the 15 patients identi¯ed using the criteria proposed above, had been diagnosed with Raynaud's syndrome.As is known, the basis of Raynaud's syndrome is a local defect in the regulation of vasomotor reactions.Thus, the revealed violation of blood °ow and an increase in spasms are only manifestations of the disease.In other patients, the haematologic abnormalities in the form of anaemia (n ¼ 5) were observed on background of the main disease.This indicates a decrease of haemoglobin and, consequently, the deterioration of blood °ow.Violation of blood °ow and angiospasm could provoke and systemic manifestations of rheumatoid disease in the form of vasculitis (n ¼ 3), which is characterized by in°ammation and destruction of blood vessels.As a result, there is a narrowing of the damaged blood vessels, violation of blood °ow and tissue destruction. 66The increase of vascular spasms could also be the result of vasodilation disorder and reduction of vasodilatory stimuli against a background of increased sympathetic nervous system activity, which is one of the possible pathogenetic factors of hypertension (n ¼ 8). 67t should be noted that some patients had the combined e®ect of several factors described.Only one patient in the diagnosis (female) did not display factors that could in°uence the functional state of the microcirculatory bed and cause increased spasms of vessels.Most likely, the detected deviation was due to the short-term increase of vasospastic activity in the background of emotional or hormonal surges, which is typical for females. 68

Conclusion
Statistically signi¯cant di®erences between the groups analyzed were found as part of the studies conducted and analysis of data received using methods of laser Doppler °owmetry, tissue re°ectance oximetry and pulse oximetry together with the cold pressor test.Wherein, the application of the proposed diagnostic criteria of tendency to angiospasm made it possible to identify subgroups in each group with the presence and absence of angiospastic disorders.In the group of 32 volunteers, 27 people were found to not display any disorders, while in the group of patients, 18 people were observed to have a relatively normal functional state of the microcirculatory bed and 15 people were observed to have a possible tendency to angiospasm.The statistical analysis of experimental data of healthy volunteers without identi¯ed angiospastic disorders and patients with identi¯ed angiospastic disorders showed that there are signi¯cant di®erences between these states, namely between a relatively normal state and presence of angiospastic disorders.Further analysis of data of these patients identi¯ed a relationship between their diagnoses and the results of laboratory studies.
Thus, the evaluation of combined noninvasive optical diagnostic method use, the cold pressor test and proposed diagnostic criteria showed a positive result.This method can be used to detect the presence of possible angiospastic disorders and related complications, as well as microcirculatory bed disorders against the background of other diseases.
Based on the above results, further experimental procedures are planned on a larger volunteer base.This would enable development of more accurate criteria for diagnostically di®erentiating disorders, i.e., explore the speci¯city and sensitivity of the methods proposed in identifying the di®erences in angiospastic disorders present in various diseases.Additionally, this would not only allow to discern the volunteer state of health or disease, but to further improve the ability to detect disease stages and their features.A more in-depth veri¯cation of the proposed methodology will also be conducted by associating the study with existing instrumental diagnosis methods.

Fig. 1 .
Fig. 1.The location of the optical sensors on the ¯ngers during the experimental studies on volunteers using the \LAKK-OP" (a), the experimental setup for the study on patients using the \LAKK-M" (b).

Fig. 3 .
Fig. 3.The results of static analysis on healthy volunteer without angiospastic disorders (No. 1) and patient with angiospastic disorders (No. 2) data: the index of blood microcirculation (a), tissue oxygen saturation (b), myogenic tone (c) and the rate of oxygen consumption (d).

Table 1 .
The main characteristics of the patient group.