Effect of whole-body cryotherapy on morphological, rheological, and biochemical blood indices in individuals with multiple sclerosis

The study aim was to examine the impact of 20 whole-body cryotherapy sessions on biochemical and rheological blood indices in multiple sclerosis individuals. The study group involved 15 women (mean age: 41.53 ± 6.98 years) with diagnosed multiple sclerosis who underwent whole-body cryotherapy sessions. The rst control group consisted of 20 women (mean age: 40.45 ± 4.77 years) with multiple sclerosis who received no cryotherapy intervention. The second control group comprised 15 women (mean age: 38.47 ± 6.0 years) without neurological diseases or other chronic conditions who participated in cryotherapy sessions. For blood indices analysis, venous blood was collected twice: on the day of cryotherapy commencement and after the 20 cryotherapy sessions. Blood counts were determined with a hematology analyzer. A laser-optical rotational cell analyzer served to investigate erythrocyte aggregation and deformability. Total serum protein was measured, and proteinogram and brinogen values were established. Statistically signicant differences were observed in red blood cells, hemoglobin, hematocrit, elongation index, total extent of aggregation, proteins (including brinogen). There was no signicant effect of the 20 cryotherapy sessions on morphological, rheological, or biochemical blood indices in women with multiple sclerosis. The intervention had a positive impact on the rheological blood properties of healthy women.


Introduction
Multiple sclerosis (MS) is a chronic disease of the central nervous system, with the key feature of demyelination foci occurrence, especially in the white matter of the brain. MS pathogenesis is not fully understood. The condition is perceived as an in ammatory demyelinating disease in which damage to the axons plays an essential role. There are no current and accurate statistical data assessing the number of people affected by MS. It is estimated that over 2,500,000 people worldwide suffer from MS, including over 630,000 in Europe, and approximately 50,000 in Poland [1,2]. Most often, the rst symptoms appear between 20 and 40 years of age [3].
MS is hypothesized to be associated with a combination of hereditary susceptibility and still undetermined environmental factors [4]. Research suggests that MS may result from a viral infection, although the potential virus has not been identi ed. Environmental factors that may be indirectly related to the disease onset are also being investigated. The thesis of a signi cant environmental impact on an increased MS risk is supported by the variable occurrence of the disease in different areas. Studies of the links between MS and the environment have pointed at the in uence of, inter alia, geoclimatic, seasonal, socioeconomic, racial, and ethnic factors [5,6]. vitamin D supplementation; consuming more than 4 cups of coffee or more than 2 alcoholic beverages per day; changing the diet immediately before or during the study; participation in other forms of physical activity directly before or during the study. The participants were included in the research program after obtaining a consent from a neurologist, a rehabilitation physician, and a physiotherapist (assessment of health status, functional status, disease stage, course, and type; determination of the applied treatment: patients with MS were treated mainly with immunomodulating agents and steroids). Body weight was assessed with a Tanita BC 418 MA device (measurement error: 0.1 kg), body height with a tape measure (measurement error: 0.5-1 cm). The Tanita BC 418 MA device also served to establish body composition with the use of the bioelectrical impedance method. The characteristics of the study group and the control groups are presented in Table 1.

Methods
Analysis of blood indices. For the analysis of blood indices, venous blood was collected twice: at baseline (study 1; on the day of whole-body cryotherapy commencement) and after the series of 20 cryotherapy sessions (study 2). In patients who did not undergo the intervention, blood was collected once (study 1). Throughout the project, blood pressure [mm Hg] was monitored in patients receiving cryotherapy: before and after each session. Fasting blood samples were collected in the morning from the basilic, cephalic, or median cubital vein into test tubes: with EDTA -for hematological analysis of whole blood; K2 potassium edetate (6 ml) was used as an anticoagulant; with clotting activator -for serum testing; the main activator ingredient is SiO 2 (6 ml). The blood was collected by a quali ed laboratory diagnostician, under the supervision of a physician, in accordance with the applicable standards of the Blood Physiology Laboratory of the University of Physical Education in Krakow. The assessment of blood indices was performed in the Blood and Skin Assessment of elongation and aggregation indices. A laser-optical rotational cell analyzer (LORCA) (RR Mechatronics, the Netherlands) was used to study erythrocyte aggregation and deformability; the results were presented as elongation and aggregation indices. The tests in the above-mentioned device were performed within 30 minutes of blood collection, at 37°C, in accordance with a standard protocol [24-26].
The blood for determining the elongation index (EI) was taken from the test tubes in the amount of 25 µl and added to 5 ml of 0.14 mM polyvinylpyrrolidone (PVP), dissolved in phosphate-buffered saline (PBS).
The test sample was placed in the measuring chamber between two concentric cylinders set in rotation. The laser light, passing through the thin layer of red blood cells suspended in the PBS solution, was de ected, giving a diffraction image on the projection screen. The EI results are provided in the range of 0.30-60.30 shear stress measured in pascals. EI is the measure of deformation of red blood cells as they move through the measurement chamber [24][25][26].
Prior to the actual aggregation test, the blood sample was oxygenated by incubation and mixing with carbogen for 15 minutes. The blood, in the amount of 1.5 ml, was introduced into the measuring chamber of the LORCA analyzer. Within 120 seconds, the cylinder was set in rotation with a shear rate of > 400 • s -1 . After 10 seconds, the centrifugation was stopped abruptly, and aggregation of red blood cells began.
The result of the computer analysis is a curve of time dependence of the scattered light intensity (for a speci c shear rate), i.e., a sylectogram [24-26].
Parameters determining the kinetics of erythrocyte aggregation were investigated: Assessment of protein level. Total serum protein was measured by using a Cobas 6000 analyzer (Roche) (biuret method). The following reaction was applied: In alkaline environment, peptide bonds of proteins form a characteristic violet-colored complex with copper ions contained in the biuret reagent. The following reagents were used: R1 -sodium hydroxide 400 mmol/l, potassium sodium tartrate 89 mmol/l; R2 -sodium hydroxide 400 mmol/l, potassium sodium tartrate 89 mmol/l, potassium iodide 61 mmol/l, zinc sulfate 24.3 mmol/l. Serum was used in the samples. The concentrations were automatically calculated by the system. The proteinogram was provided by a Minicap (Sebia) analyzer and the capillary method was applied. the albumin-globulin (A/G) ratio.
Determination of brinogen concentration. For the determination of brinogen concentration, 50 µl of plasma was added to 100 µl of thromboplastin with calcium chloride. The result is based on the measurement of the time from reagent addition to clot formation; then, the result is converted to g/l. The assessments were performed with a Bio-Ksel Chrom-7 camera (spectrophotometric method).
Description of the intervention. The whole-body cryotherapy sessions were carried out in the Malopolska Cryotherapy Rehabilitation Center in Krakow. The applied cryochamber temperatures were as follows: vestibule temperature: -60°C; chamber temperature: -120°C.
The time of a single whole-body cryotherapy session in the groups who underwent the intervention in the study was as follows: 1.5 minutes (the rst session); 2 minutes (the second session); 3 minutes (the third session on).
One session per day was performed, 5 times a week (every day in the same time period of 3:00-5:00 p.m.). Overall, there were 20 sessions.
Women entering the cryochamber were dressed in swimsuits, high woolen socks, and shoes with a high wooden sole. The parts of the body most exposed to low temperature were covered (the subjects wore gloves, a head covering, and a face mask). After each cryotherapy session, the patients warmed up on a Kettler Corsa cycle ergometer without any resistance for 15 minutes. The sessions were directly supervised by a trained physiotherapist. A physician was present at the Rehabilitation Center throughout the entire duration of each session.
During the procedure, visual contact with the patients was maintained through thermal windows; additionally, the interventions were monitored with a camera and the patients were given information on the current session time (every 30 seconds). The cryochamber is equipped with a visible alarm button, as well as electronic systems monitoring the oxygen level, humidity, and temperature of both the vestibule and the proper chamber.
Statistical analysis. Data are presented as mean values and standard deviations. The normality of distributions was veri ed with the Shapiro-Wilk test. The differences between the study group and the control groups were assessed by using the one-way analysis of variance (ANOVA) or, if its assumptions were not met, the Kruskal-Wallis test. For post-hoc evaluation, Tukey's test for unequal sample sizes was applied, or, respectively, the multiple comparison test of mean ranks for all Dunn's trials. The dependent variables were compared with Student's t-test for related variables or, if its assumptions were not met, with the Wilcoxon test. Independent variables were compared with the Mann-Whitney U test. The signi cance level of α = 0.05 was adopted in the analyses. The applied tests veri ed two-sided hypotheses. The analyses were performed with the use of the Statistica 13 package (Tibco Software Inc., USA).
There was also a signi cantly higher mean baseline HGB level in the group of healthy women (CONTROL-CRYO) compared with patients with MS (CRYO-MS) before whole-body cryotherapy (P = 0.045). After the intervention, a statistically signi cantly (P = 0.014) lower mean HGB level (3.81%) was noted in the group of healthy women.
As for the EI, a signi cant difference was observed in the baseline values at the shear stress of 4.24-60.30 Pa: EI was lower in healthy women (CONTROL-CRYO) as compared with MS women who received (CRYO-MS) and who did not receive whole-body cryotherapy (CONTROL-MS) (P ≤ 0.016). After the intervention, an increase in the EI value was noted in healthy women at the shear stress of 2.19-60.30 Pa. The average level of the index variability was 28.07%.
A statistically signi cantly higher baseline AMP was determined in healthy women (21.62 ± 4.06 a.u.) than in MS controls who received no cryotherapy (18.16 ± 4.57 a.u.). There were no statistically signi cant changes in AMP after the intervention.
When analyzing protein levels, lower baseline values of total protein were observed in the groups of MS patients (70.17 ± 3.98 g/l and 70.29 ± 2.79 g/l in the CRYO-MS and CONTROL-MS group, respectively) compared with healthy women (73.56 ± 3.68 g/l). There was also a statistically signi cant reduction (by 2.49%) in the total protein level in healthy women after whole-body cryotherapy application (71.73 ± 2.53 g/l). Mean albumin levels slightly decreased in healthy women after the intervention (CONTROL-CRYO) (P = 0.009). There were no statistically signi cant changes in albumin levels after the use of whole-body cryotherapy. MS patients presented lower baseline levels of gamma globulins (9.41 ± 2.22 g/l and 9.47 ± 2.29 g/l in the CRYO-MS and CONTROL-MS group, respectively) than healthy patients (11.53 ± 2.37 g/l); the average difference equaled 18.13%. There were no statistically signi cant changes after whole-body cryotherapy application.
With reference to the mean levels of brinogen, a statistically signi cant increase was observed in healthy women after the cryotherapy intervention (34.13% in the CONTROL-CRYO group), as well as a statistically insigni cant increase, owing to a large discrepancy, in patients with MS who received wholebody cryotherapy (44.24% in the CRYO-MS group). There were no statistically signi cant between-group differences in baseline brinogen levels.
Detailed results are presented in Figures 2 and 3 and Tables 2-5.

Discussion
Because of the lack of an unambiguous answer as for MS etiopathogenesis and, consequently, the limited options of causal treatment, no effective therapy has been developed to combat the disease [4]. The currently available interventions allow to reduce the disease activity, inhibit its progression, modify the autoimmune process, in uence the intensity of relapses, and alleviate or treat the concomitant clinical symptoms. MS therapy is a di cult process, which is related, inter alia, to the multitude of symptoms and their overlapping [27,28]. Despite the increasingly detailed knowledge about the disease pathomechanism, the pharmacological treatment options that might be widely applied are quite limited. Therefore, symptomatic treatment, including physical activity and physiotherapy, plays an important role besides pharmacotherapy [29,30].
Rheology investigates blood ow through blood vessels. It concerns whole blood, as well as plasma and morphotic elements, especially red blood cells [31]. The basic factors that in uence blood ow involve RBC, red blood cell deformability and aggregation, plasma viscosity, and HCT [31-33].
The phenomenon of erythrocyte deformability plays an important role in the ow of blood cells through capillaries with a diameter even two times smaller than the cells [34]. Normal erythrocytes deform under stress mainly because they do not have cell nuclei, their cytoplasm has a relatively low viscosity, the cell membrane presents favorable viscoelastic properties, and the appropriate shape ensures a high ratio of free surface to volume [26]. Changes in the blood cell shape depend on the quality of the spectrin-actin network in connection with calcium and ATP ions, and the reasons for this capacity decrease mainly involve the cell age, mechanical damage, and disease factors [10,35].
According to Maeda [36], appropriate erythrocyte deformability plays a key role in the blood ow in the vascular system. In turn, the appropriate erythrocyte shape, intracellular viscosity, and the cell membrane wall stiffness depend on the HGB level, which signi cantly in uences the deformation capacity. Red blood cell deformability is crucial as it allows the cells to pass through capillaries. The lower the deformability, the higher the blood viscosity and the worse the blood ow in the microcirculation [36].
Spontaneous erythrocyte aggregation in whole blood, i.e., the formation of three-dimensional erythrocyte structures, is a reversible physiological phenomenon that plays a key role in blood ow at low shear rates and signi cantly increases blood viscosity [26]. Erythrocyte aggregation is a complex dynamic phenomenon, activated by various factors, especially plasma-related ones, and pathological processes. Physiological shear stresses are to prevent aggregate formation, and the absence of shear stresses may be among the many causes of cardiovascular disease [37].
The degree of red blood cell aggregation depends on the ow conditions, properties of cell membranes, and physicochemical properties of cells [33]. Small blood vessels, where the shear rates are usually low, are particularly susceptible to the formation of erythrocyte aggregates. This ultimately causes a decrease in blood ow velocity, or even its inhibition, and, as a consequence, under-oxygenation of cells and The hematological symptoms in MS patients are unspeci c. A homogeneous mechanism of their occurrence is di cult to determine and very likely not to exist at all. It has been suggested that hematological changes in MS may be related to such factors as complex humoral and cellular response, biochemical disorders, changes related to the attachment and transport of vitamin B12, the applied pharmacological treatment, and the phase of the disease (relapse or remission) [39][40][41].
Erythrocyte elasticity is signi cantly in uenced, inter alia, by intracellular viscosity, affected by HGB. Appropriate MCH and MCHC values determine the potential for erythrocyte deformability even in cases of hyper-or hypotonic changes in the blood environment [31]. Najim al-Din et al. [42] did not nd signi cant HGB level differences between MS patients and those with other neurological diseases; they also con rmed the thesis by Reynolds and Linnell [41] about the tendency for macrocytosis without concomitant anemia in MS patients. In the presented study, a statistically signi cantly higher mean baseline level of HGB was observed in the group of healthy women compared with the mean level in MS patients before whole-body cryotherapy (P = 0.045). After the intervention, a statistically signi cantly lower mean HGB level was noted in healthy women (P = 0.014). The analysis showed no statistically signi cant differences in MCH after whole-body cryotherapy application or at baseline. However, there was a statistically signi cant minimal increase in MCHC in healthy women after whole-body cryotherapy application (P = 0.013).
Grasso et al. [43] found no pathological HGB or RBC values in MS patients. They performed blood tests in MS patients (EDSS score: 1-9) and observed no difference in MCV as compared with the control group. At the same time, as the comparative group consisted of individuals who were not completely healthy, the results were also referred to the assumed physiological norms. Notably, the MCV results exceeded the assumed norms in one person from the study group and one from the control group only. No correlation was established between the patient's functional status expressed in the EDSS score and the MCV value.
In subsequent studies, Kocer et al. [44] also failed to con rm macrocytosis in MS patients examined during a relapse. In their research, MCV remained within the normal range in over 77% of the participants, while microcytosis was observed in the others. De Freitas et al. [45] also found no differences in MCV between MS patients undergoing steroid therapy and those who received no pharmacological treatment.
In the present study, there were also no statistically signi cant WBC changes in MS patients compared with the control group. However, a statistically signi cant difference was observed in the baseline RBC levels, which were lower in patients with MS (CRYO-MS and CONTROL-MS groups) than in healthy women (CONTROL-CRYO group). Baseline MCV did not differ between groups, with a slight MCV reduction observed in healthy women after whole-body cryotherapy application (P = 0.028).
The red blood cell distribution width (RDW), a measure of erythrocyte size and volume, affects deformability. A study found an increased RDW in patients with relapsing-remitting MS compared with healthy controls, which may underlie the altered red blood cell deformability in MS. Furthermore, increased RDW was observed to be positively associated with EDSS scores. A study that involved 109 MS patients and 130 healthy individuals failed to control other potential confounding factors affecting RDW and lacked adequate pathological controls. More research is needed on the use of RDW as a biomarker [46]. Morphological changes in erythrocytes (macrocytes and echinocytes) are positively correlated with MS severity and may impair red blood cell deformability [47].
Platelets also play an important role in the coagulation cascade; they are abundant in MS [48]. Platelets themselves do not directly affect erythrocyte aggregation, but in uence thrombotic processes [49,50], which supports the idea of ischemic tissue damage [51]. In the present study, no statistically signi cant changes in PLT were established after whole-body cryotherapy application or at baseline.
Brunetti et al. [39] observed increased values of whole blood viscosity indices, with coexistent normal levels of plasma viscosity and HCT. They suggested that the raised blood viscosity was due to a decrease in erythrocyte deformability. Later studies by Pollock et al. [40] did not con rm this hypothesis, showing no visible differences in erythrocyte deformability between MS patients and healthy controls. Research related to the morphology of erythrocytes was also carried out by Simpson et al. [52], who reported that MS patients presented a lower level of sphingomyelin in the erythrocyte cell membrane as compared with the control group. They demonstrated impaired erythrocyte deformability, which can increase blood viscosity in MS patients. The authors observed an increase in MS patients' blood viscosity that varied depending on sex. In women, the values of blood viscosity were signi cantly higher than in the control group at the shear rates of 1 • s − 1 , 10 • s − 1 , and 100 • s − 1 , while in men, blood viscosity was statistically signi cantly higher only at one shear rate (1 • s − 1 ). Ernst [53] noted, however, that changes in viscosity indices did not always correlate with the level of erythrocyte deformability, as they were also in uenced by the administered steroid therapy. This was con rmed by de Freitas et al. [45], who investigated the effect of steroid therapy on red blood cell membrane stability. They found that erythrocyte membranes in MS patients were less resistant to damaging factors than erythrocyte membranes in healthy individuals, while steroid therapy improved erythrocyte membrane resistance and brought it closer to that observed in healthy people.
When analyzing the level of HCT in the conducted study, a statistically signi cant difference was observed in the baseline values: these were lower in MS patients receiving the intervention (35.97 ± 5.65%) and in the MS control group (35.90 ± 5.88%) compared with healthy women (40.54 ± 2.50%). Moreover, healthy women were characterized by a statistically signi cantly lower HCT level after wholebody cryotherapy application (P = 0.003). In turn, with reference to EI, a statistically signi cant difference was demonstrated in baseline values at the shear stress of 4.24-60.30 Pa: the result was unexpectedly lower in healthy women compared with those with MS who received or who did not receive cryotherapy (P ≤ 0.016). In healthy women, a favorable increase in EI was noted at the shear stress of 2.19-60.30 Pa after the intervention. The average level of EI variability equaled 28.07%. The application of whole-body cryotherapy signi cantly increased erythrocyte deformability and decreased HCT values in healthy women, which positively in uences their rheological blood properties.
Kowal and Marcinkowska-Gapińska [54] published the preliminary results of their research on the comparison of hemorheological properties in MS patients and individuals with acute cerebral ischemia.
They observed a statistically signi cantly higher HCT value and a lower measure of red blood cell aggregation capacity in patients with MS compared with those after an acute cerebral ischemia incident. The differences in erythrocyte stiffness and the tendency to aggregation were not statistically signi cant.
In the study, however, MS patients constituted a very small group, and the research was not continued among a wider MS cohort. The mean AI values in the study group and in the control group were not signi cantly different before and after whole-body cryotherapy. There were also no statistically signi cant changes in baseline AI or T1/2 values.
Fibrinogen is a 340-kDa glycoprotein synthesized in the liver, with a plasma concentration of approximately 150-400 mg/dl. The protein is involved in blood clotting and hemostasis, as well as in in ammation and tissue repair. Fibrinogen facilitates platelet aggregation by glycoprotein IIb/IIIa receptor binding and forming a brin monomer that rapidly polymerizes to create a clot [55,56]. With in ammatory response, plasma brinogen levels rise 2-3-fold, leading to cell aggregation and an increase in blood viscosity [57]. Studies have shown that brinogen can modulate in ammatory response by leukocyte activation and synthesis of pro-in ammatory mediators (cytokines and chemokines) [55,58]. Fibrinogen is not a clear indicator of the blood-brain barrier disruption, but it does activate glial cells, which results in blood-brain barrier dysfunction in MS patients [59].
Acuña et al. [60] demonstrated that high plasma brinogen levels (> 417 mg/dl) were associated with active changes in magnetic resonance imaging in MS patients during relapses. In an earlier study on brinogen in MS patients, Ehling et al. [61] showed no elevation of brinogen in the cerebrospinal uid or plasma, probably because they compared brinogen levels between MS patients and those with central nervous system infections; in addition, less than a third of MS patients experienced an acute relapse at the time of brinogen analysis, with the rest presenting an inactive disease or a chronic progressive course.
Despite the in ammatory events, it seems that patients with relapsing-remitting MS do not have elevated levels of brinogen in remission [62]. However, D-dimer levels are elevated [63] and low brinogen levels during remission (MS patients vs. controls) do not rule out brinogen increase during relapse, especially if one considers the role of brinogen in MS pathology, where brin is involved in cytokine release and microglia activation in the central nervous system [64,65]. When analyzing the mean levels of brinogen in the present study, no increased values were observed in women with MS, while after the application of a series of 20 whole-body cryotherapy sessions, there was a statistically signi cant rise in healthy women (by 34.13%) and a statistically insigni cant rise (owing to the large discrepancy) in MS patients (by as much as 44.24%).
In clinical practice, plasma brinogen may be a valuable and easy biomarker of activity during relapses in MS patients, but prospective studies in larger groups are needed to con rm these results.
It has been found that although all MS patients show an increase in oxidative damage biomarkers, there seems to be no correlation between the degree of the increase and the disease severity. It is now suggested that oxidative changes are not accompanied by in ammatory activity in these patients as measured by changes in WBC and C-reactive protein levels. This would mean that oxidative stress precedes the in ammatory response, which would indirectly support the hypothesis that oxidative stress alters blood-brain barrier permeability and stimulates monocyte adhesion to vascular endothelium [66]. Erythrocytes may contribute to the pathophysiological mechanisms of MS through impaired antioxidant capacity and altered hemorheology, leading to an increase in oxidative stress and to potential ischemic tissue damage, respectively [67].
Studies on the effect of whole-body cryotherapy in MS patients were also conducted by Bryczkowska et al. [68]. In healthy individuals, the rst changes in the lipid pro le were observed after 20 daily whole-body cryotherapy sessions [20], while in MS patients, after a series of 30 whole-body cryotherapy sessions, researchers did not report signi cant changes in total protein, albumin, uric acid, and glucose concentrations or in the lipid pro le [68]. In the present study, lower baseline total protein levels were demonstrated in the groups of MS patients (70.17 ± 3.98 g/l and 70.29 ± 2.79 g/l) compared with healthy women (73.56 ± 3.68 g/l); there was also a statistically signi cant reduction of the parameter among healthy women after whole-body cryotherapy application (2.49%). No signi cant differences were indicated in the other proteinogram indices, i.e., albumin, alpha-1 globulins, alpha-2 globulins, beta-1 globulins, beta-2 globulins, except for lower baseline levels of gamma globulins in the groups of MS patients compared with healthy women (by 18.13% on average).
Studying the role of erythrocytes in MS may reveal further speci c differences that could be used as the disease biomarkers, as well as broaden our understanding of the pathological mechanisms of this complex and heterogeneous disease. This, in turn, could lead to the discovery of new and innovative therapeutic targets, thus signi cantly improving patients' quality of life.
Summing up, in the light of the available literature and the results of own research, it can be concluded that whole-body cryotherapy is an effective method to combat or inhibit the progress of many diseases and their negative consequences, thus contributing to maintaining the best possible body tness. The presented study is probably the rst one to assess the in uence of whole-body cryotherapy on rheological blood properties, including EI and AI, in MS women. No pathological or harmful changes were observed after whole-body cryotherapy application. In healthy participants, the intervention resulted in improved erythrocyte exibility, which adds to better cell oxygenation.

Study Limitation
The study is not without limitations. An important aspect that could have affected the trial results is a relatively small number of individuals in the study groups. The obtained results are important for determining the safety of whole-body cryotherapy in the eld of clinical trials, and the research should be continued (with randomization) in larger and more diverse groups of patients.     Table 5. Assessment of therapeutic intervention in the CONTROL-CRYO group. MS multiple sclerosis, WBC white blood cell, RBC red blood cell, HGB hemoglobin, HCT hematocrit, PLT platelet, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, EI elongation index, AI aggregation index, AMP amplitude (total extent of aggregation), T1/2 half time kinetics of aggregation.