Abnormal rheological properties of red blood cells as a potential marker of Gulf War Illness: A preliminary study

Abstract

BACKGROUND:

Veterans with Gulf War Illness (GWI) experience chronic symptoms that include fatigue, pain, and cognitive impairment. This symptom cluster may be the consequence of impaired tissue oxygen delivery due to red blood cell (RBC) dysfunction.

OBJECTIVE:

The purpose of this preliminary study was to determine whether the microrheological behavior of RBCs is altered in GWI.

METHODS:

We recruited 17 cases of GWI (GWI+) and 10 age matched controls (GWI–), and examined RBC deformability and aggregation via ektacytometry along with measurement of complete blood counts.

RESULTS:

RBCs were more deformable in GWI+, as indicated by higher elongation indices particularly at higher shear stress values (5.33, 9.49, and 16.89) when compared to GWI–. Aggregation formation, stability and kinetics were similar between GWI+and GWI–. Complete blood counts were also similar, with the exception of mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and RBC distribution width (RDW) which was elevated in GWI+.

CONCLUSIONS:

In this preliminary study, we observed increased deformability along with increased MCH, MCHC and RDW in veterans with GWI+, which may contribute to the symptomatology of GWI. Further research is required to confirm our findings and the role of RBC microrheology in GWI.

Keywords

Persian Gulf Syndrome hemorheology rheology fatigue blood cell count

1 Introduction

Currently, 25–32% of military personnel that were deployed to the Persian Gulf for Operations Desert Shield and Storm (1990-1991) are afflicted with the chronic disorder known as Gulf War Illness (GWI) [49]. Veterans with GWI exhibit persistent health issues such as unremitting fatigue, widespread musculoskeletal pain, and cognitive impairment being the most commonly reported symptoms [13 , 46]. GWI shares similarities with other illnesses of unknown origin such as chronic fatigue syndrome [31], and these symptom clusters may be the consequence of reduced blood flow and impaired tissue oxygen delivery [4, 32]. Reduced blood flow in both chronic fatigue syndrome and GWI has been attributed, in part, to abnormal autonomic control of the vasculature [4 , 32]. However, less attention has been paid to the intrinsic properties involved with blood flow regulation.

Blood flow is constantly adapting to meet the metabolic demands of a given tissue, and this response involves both a vascular component as well as the biomechanical or rheological properties of blood. Rheological properties of blood are influenced by the viscosity, shape, deformability, and aggregation of red blood cells (RBC) as well as their degree of adhesion to endothelial cells [3]. The ability of RBCs to successfully traverse the microvasculature is a critical factor in respiratory gas exchange. Only two studies have considered the fluidity of blood in GWI, and both reported increased platelet activation [18, 20]. Despite similarities, the interpretations of these studies differed. Hannan et al. [18] suggested that observed platelet and coagulation abnormalities were the result of an abnormal immunological response to undefined antigen(s), in support of related literature on immune dysregulation in GWI [7 , 50]. However, the studied patients in this sample were said to have underlying conditions (i.e., hereditary thrombophilia and hypofibrinolysis) which may affect this interpretation. More recently, Johnson et al. [20] reported higher platelet aggregation in veterans with GWI, particularly in those individuals with higher levels of C-reactive protein. Greater platelet aggregation may be caused by elevated C-reactive protein in support of an underlying chronic inflammatory state as a contributing factor in the pathogenesis and symptomology of GWI [19]. Beyond these two studies, there is a paucity of data regarding rheological properties of RBCs in GWI, which is surprising given the well-known impact of impaired hemorheology on blood fluidity and the resultant impact on tissue perfusion and function [3].

We hypothesize that symptoms of GWI are manifested secondary to impairments in the rheological properties of RBCs, and that a better understanding of the role of the intrinsic properties of blood in these patients is relevant for potential therapies to improve hemorheological properties. Therefore, the objective of this study was to evaluate the rheological properties of RBCs, including deformability and aggregation, in veterans with GWI.

2 Methods

2.1 Study design and participants

This cross-sectional study was approved by the Veterans Affairs New Jersey Health Care System’s Institutional Review Board, and all subjects gave written informed consent. Study participants included 17 GWI cases (GWI+) and 10 controls (GWI–), with cases defined in accordance to the Kansas criteria for GWI [42]. Veterans with GWI+ were required to have moderately severe symptoms in at least 3 of 6 symptom domains (i.e., fatigue, pain, neurocognitive, gastrointestinal, skin and respiratory) for at least the past 6 months that could be not be explained by concurrent comorbid conditions (e.g., diabetes, heart disease, cancer, etc.). Medical histories were obtained through interview, questionnaires and computerized medical record review. Demographics and clinical characteristics for GWI+ and GWI–are provided in Table 1.

Table 1
Baseline characteristics

GWI– (n = 10) GWI+ (n = 17)

Demographics

Age (years) 52.3±1.9 49.3±1.22

Height (cm) 174.1±2.46 174.8±1.60

Weight (kg) 95.2±5.36 90.0±3.62

Body mass index (kg/m²) 31.6±1.88 29.4±0.97

Tobacco use (pack-years) 10.3±4.25 11.1±3.40

Vital Signs

Physical activity (min wk^–1) 154.3±61.21 103.2±48.12

Resting heart rate (bpm) 69.7±4.29 70.4±2.78

Resting mean blood pressure (mmHg) 90.8±2.98 88.9±1.56

Oxygen saturation (%) 97.2±0.59 97.1±0.38

Symptom questionnaires

Fatigue severity scale 26.3±4.63 47.1±3.29^‡

VR-36 physical composite score 52.5±3.67 38.6±2.78^‡

Kansas GWI questionnaire 8.7±3.36 38.1±3.98^‡

Complete blood count

WBC (10^–3/μl) 7.0±0.51 6.1±0.62

RBC (10^–6/μl) 4.9±0.11 4.5±0.18

Platelets (10^–3/μl) 182.6±16.52 160.4±15.53

Hb (g/dl) 14.0±0.39 13.8±0.53

Hct (g/dl) 41.5±1.08 39.8±1.64

MCV (fl) 85.6±2.24 87.7±1.11

MCH (pg) 28.9±0.82 30.5±0.38*

MCHC (g/dl) 33.8±0.23 34.8±0.32*

RDW (%) 14.6±0.56 16.4±0.59*

	GWI– (n = 10)	GWI+ (n = 17)
Demographics
Age (years)	52.3±1.9	49.3±1.22
Height (cm)	174.1±2.46	174.8±1.60
Weight (kg)	95.2±5.36	90.0±3.62
Body mass index (kg/m²)	31.6±1.88	29.4±0.97
Tobacco use (pack-years)	10.3±4.25	11.1±3.40
Vital Signs
Physical activity (min wk^–1)	154.3±61.21	103.2±48.12
Resting heart rate (bpm)	69.7±4.29	70.4±2.78
Resting mean blood pressure (mmHg)	90.8±2.98	88.9±1.56
Oxygen saturation (%)	97.2±0.59	97.1±0.38
Symptom questionnaires
Fatigue severity scale	26.3±4.63	47.1±3.29^‡
VR-36 physical composite score	52.5±3.67	38.6±2.78^‡
Kansas GWI questionnaire	8.7±3.36	38.1±3.98^‡
Complete blood count
WBC (10^–3/μl)	7.0±0.51	6.1±0.62
RBC (10^–6/μl)	4.9±0.11	4.5±0.18
Platelets (10^–3/μl)	182.6±16.52	160.4±15.53
Hb (g/dl)	14.0±0.39	13.8±0.53
Hct (g/dl)	41.5±1.08	39.8±1.64
MCV (fl)	85.6±2.24	87.7±1.11
MCH (pg)	28.9±0.82	30.5±0.38*
MCHC (g/dl)	33.8±0.23	34.8±0.32*
RDW (%)	14.6±0.56	16.4±0.59*

Demographics, vital signs, symptom questionnaires and complete blood count results are presented as mean±SE. WBC: white blood cells; RBC: red blood cells, Hb hemoglobin; Hct: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; RDW: red blood cell distribution width. *p < 0.05; ^‡p < 0.01.

2.2 Symptom questionnaires

All participants completed the Kansas GWI screening questionnaire [42], fatigue severity scale [26], and the Veterans version of the Short Form 36 Health Survey (VR-36) [47]. The Kansas GWI screening questionnaire is a 29-item Likert-style questionnaire used primarily to assign case status (GWI+ or GWI–), but we also used this questionnaire to derive an estimate of GWI severity. For each item, participants may indicate whether a symptom is absent (score of ‘0’) or present to mild, moderate, or severe severity (scores of ‘1–3’, respectively). We computed a summed score (range: 0 – 87) as an index of GWI severity with higher scores indicating greater symptom severity. The fatigue severity scale is comprised of 9-items to quantify fatigue severity and its impact on self-reported activity (range: 0–63), with higher scores indicating greater fatigue severity. From the VR-36, we computed a physical composite score to provide an index of physical health-related functioning that was standardized to a population mean of 50 and standard deviation of 10, with higher scores reflecting better overall health [21 , 47]. Questionnaire responses are provided in Table 1.

2.3 Blood sampling

Blood samples were drawn from the antecubital vein in sterile vacutainers containing sodium citrate (3.2%). All subsequent measurements were completed within 2 hours after sample collection. The blood samples were used to study RBC deformability and aggregation. A complete peripheral blood count was also performed using an electronic hematology analyzer (Sysmex XE-5000; Sysmex America, Inc. Lincolnshire, IL) to derive an estimate of white blood cells (WBC), RBC, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and RBC distribution width (RDW). Values are shown in Table 1.

2.4 Erythrocyte deformability measurements

RBC deformability (i.e., the ability of the entire cell to adopt a new shape when subjected to mechanical forces) was determined at 37C at various fluid shear stresses by laser diffraction analysis using an ektacytometer (LORRCA MaxSis, RR Mechatronics; Netherlands) as described elsewhere [2]. Briefly, immediately after collection, venous citrated blood was mixed with an isotonic viscous solution (0.14 mM polyvinylpyrrolidone, osmolality 316 mOsm/Kg, viscosity 28.7mPa at 37C), and sheared in a Couette system composed a glass cup and a precisely fitting bob, with a gap of 0.3 mm between the cylinders. A laser beam is directed through the sheared sample, and the diffraction pattern produced by the deformed RBCs is analyzed by microcomputer. On the basis of geometry the elliptical diffraction pattern, an elongation index (EI), is calculated as; EI=(L-W)/(L + W), where L and W are the length and width of the diffraction pattern, respectively. EI values were determined from nine shear stresses between 0.3 and 30 Pa, and the shear stress required for half-maximal deformation (SS_1/2) and maximal EI (EI_max) [2] were automatically calculated by the ektacytometer software.

2.5 Erythrocyte aggregation measurements

RBC aggregability was also determined using the same ektacytometer (LORRCA MaxSis). For this analysis, we transferred 2 ml of whole blood to 25 ml erylermyer flasks and mixed on an orbital shaker for at least 15 minutes to ensure blood was fully oxygenated, and 1 ml of oxygenized whole blood was placed into the gap between the cylinders. The samples were initially sheared at 500 s^–1 to disperse pre-existing aggregates, following which the shear is abruptly reduced to zero. RBC aggregation parameters were determined with a syllectogram, which is a curve illustrating the change in the light intensity of scattered light during 120 sec corresponding to the progress of aggregation [2]. In this study, the following aggregation parameters were used: 1) amplitude (AMP, the total extent of aggregation), 2) aggregation half time (T_1/2, time that elapses until the peak intensity is reduced by half, reflects the kinetics of aggregation), 3) aggregation index (AI, a larger index of aggregation and/or a greater aggregation amplitude), and 4) γ at dIsc min (minimal shear rate required to disperse RBC aggregates, reflects aggregate stability).

2.6 Statistical analysis

Results are presented as the mean±SE. Between-group comparisons were performed using repeated-measures (i.e., multiple shear stresses) ANOVA. Unpaired t-tests were used to compare demographics questionnaire responses, and complete blood counts. Spearman rank correlations were used to assess relationship between blood indices and questionnaires. Statistical significance was set at an alpha of 0.05 and tests were conducted in SPSS (v24.0).

3 Results

3.1 Participants

Participant demographics are provided in Table 1. GWI+ and GWI–groups were for basic demographic variables and vital signs. Consistent with case assignment, veterans with GWI+ reported greater fatigue severity, poorer physical health-related functioning, and greater GWI severity (Table 1).

3.2 Complete blood counts

Complete blood counts were obtained for all participants except for one case (GWI+) that was unable to be processed; therefore, data were available for 16 of 17 cases. Blood count parameters (see Table 1) were similar between groups, except for MCH (p = 0.05), MCHC (p = 0.03), and RDW (p = 0.05). Bivariate associations were observed between MCH and the physical composite score (ρ= –0.40, p = 0.02) and the summed Kansas GWI score (ρ= 0.40, p = 0.02). MCV was also associated with the physical composite score (ρ= –0.40, p = 0.03), but not with the summed Kansas GWI score (ρ= 0.30, p = 0.08). See Fig. 2 for 3D scatter plots. No other significant associations were observed for any other parameter.

3.3 Erythrocyte deformability

One control sample (GWI–) was unable to be processed leaving 9 of 10 controls for analysis. The relationship between RBC deformability and shear stress between GWI+ and GWI–was typically sigmoidal and EI increased with the rise in shear stress (Fig. 1). Between (p = 0.02) and within-group effects (p < 0.001) were significant, but an interaction was not detected. Follow-up analyses indicated higher EI’s at several shear stresses (Fig. 1) for GWI+ in comparison to GWI–. EI_max was significantly higher in GWI+ (GWI–, GWI+: 0.56±0.01, 0.60±0.01; p = 0.02; Fig. 3), but the SS1/2 to EI_max ratios were similar between groups (GWI–, GWI+: 3.31±0.28, 3.21±0.28; p = 0.83). No significant associations were observed between deformability measures and symptom questionnaires.

Fig.1

Shear stress-elongation index (EI) curves. Elongation indices (EI) at corresponding shear stresses representing red blood cell deformability are presented as mean±SE for each group. Filled (•) and open (∘) circles represent GWI+ (n = 16) and GWI–(n = 9), respectively. *p < 0.05.

Fig.2

Relationship between RBC indices and symptoms. 3D scatter plots were used to illustrate the relationship between (A) MCV and (B) MCH with the VR-36 physical composite score (‘physical health’) and summed Kansas GWI score (‘GWI severity’).

Fig.3

Maximal RBC deformability. Dot density plot of maximal elongation index for GWI–(open circles) and GWI+ (shaded circles). Horizontal bar represents the mean, and groups were significantly different (p = 0.020).

3.4 Erythrocyte aggregation

Aggregation indices were not available for 6 cases (GWI+) and 3 controls (GWI–) due to technical error. Aggregate formation (GWI–, GWI+; AI: 59.46±2.76, 61.44±2.05 and AMP: 37.51±3.04, 38.27±1.48), and the kinetics (T_1/2: 2.83±0.39, 2.58±0.23) and stability (γ at dIsc min: 190.89±33.3, 159.55±12.37) of aggregate formation were similar between groups. No significant associations were observed between deformability measures and symptom questionnaires.

4 Discussion

This is the first study to examine the interrelation of Gulf War Illness (GWI) with red blood cell (RBC) rheology. Our results show that RBCs from veterans with GWI are more deformable than RBCs obtained from age- and sex-matched controls. This phenomenon of enhanced RBC deformability (i.e., increased EI) in GWI+ relative to controls (GWI–) was observed primarily at higher values of shear stress (Fig. 1), which suggests differences in internal viscosity rather than membrane elasticity is determining the enhanced deformability. This interpretation is further supported by hematological indices namely, the larger MCH, MCHC and RDW% in GWI+ group. Overall, our findings suggest abnormal rheological properties of RBCs are characteristic of veterans with GWI.

Our findings may appear paradoxical as increased deformability is generally thought to confer beneficial effects on the circulation [33]; however, Bor-Kucukatay et al. [5] have previously noted that if RBCs become too deformable, pulmonary capillary transit times are accelerated thereby resulting in less binding of oxygen. Further, there are other reports in the literature of maladaptive increased RBC deformability. For example, Vaya et al. [44] reported that patients with macrocytosis (i.e., enlargement of RBCs) showed significantly higher EI’s than normocytic controls at high shear stresses, suggesting that higher EI’s are a consequence of increased RBC size. RBC size is also indicative of the age of the cell, and RBCs in circulation are heterogeneous consisting of both young and mature cells. There are several lines of evidence demonstrating that circulating blood of elderly individuals is comprised primarily of younger cells that are both larger and lower in density [12, 38], consistent with an earlier release of cells from the bone marrow. In addition to greater deformability, younger RBCs in circulation have also been shown to be associated with memory impairments in elderly individuals [11 , 16] as well as in women during late pregnancy [30], even after accounting for potential confounders. Although the present study was not designed to evaluate cognitive function, cognitive impairment is a hallmark symptom of GWI as problems with memory [8] and executive function [8 , 43] are well described. Future studies should consider exploring whether larger more deformable cells in GWI is also associated with cognitive impairment.

Consistent with more deformable RBCs in GWI, we also observed increased red blood cell distribution width (RDW), which provides a measure of RBC volume variation (i.e., anisocytosis). RDW is a strong risk factor for increased all-cause cardiovascular mortality in patients with heart failure [22 , 40] and in the general population [6 , 37]. Higher RDW levels have also been shown to be associated with impaired exercise capacity in patients with chronic disease [10] and in healthy middle-aged adults [14], which support exercise-induced symptoms and exercise intolerance commonly reported in GWI. Based on our observed findings of greater deformability and anisocytosis, we speculate that abnormal rheological properties of RBCs in GWI could contribute to exercise-induced symptoms. This supposition is reinforced by the fundamental role of hemorheology on blood flow and the efficient transport and delivery of oxygen [1].

Our study is not without limitations. First, we evaluated RBC dynamics at rest and without provocation of symptoms, as commonly achieved with physical exercise. The effects of exercise on hemorheology is an area of great interest as well as clinical importance [9]. As 96% of veterans with GWI endorse post-exertional malaise [24], exploring the relationships between hemorheological and metabolic responses to exercise and recovery in GWI is deserving of attention. Second, our sample is comprised only of male participants, which given noted sex differences in rheological properties of blood [23], future studies should also include female veterans with GWI. Lastly, as this study was designed to establish proof-of-concept of whether rheological properties are disturbed in GWI, we can only speculate on the specific mechanisms that may contribute to our observed findings of increased deformability. For example, many of the symptoms reported by veterans with GWI can be interpreted to indicate cholinergic dysfunction [17], which may be another possible explanation for the observed increased RBC deformability. Specifically, Muravyov et al. [34] showed RBC deformability increased following exposure to either catecholamines or acetylcholine. Again, this is only conjecture since cholinergic effectors were not measured during this study. Other factors related to deformability, such as fibrinogen levels or whole blood viscosity, could also not be elucidated. The present study is hypothesis-generating and further research with a larger number of participants is required to confirm our findings.

In summary, the present study is the first to focus on RBC deformability and aggregation characteristics in GWI veterans and age-matched controls at rest in an attempt to define the role of RBCs in the pathophysiology of GWI. Although aggregation parameters were similar between veterans with and without GWI, the GWI veterans were marked by increased RBC deformability at rest compared to controls. The altered deformability along with increased MCH, MCHC and RDW may play a role in the clinical status (fatigue) of veterans with GWI. Nevertheless, further studies are clearly warranted to better understand the relationship between RBC deformability and aggregation in the clinical expression of fatigue in GWI veterans, which could aid in defining more accurately the role of blood rheology in the pathology of GWI.

Footnotes

Acknowledgments

The authors gratefully acknowledge the volunteers who participated in this study. This work was supported by the Department of Veterans Affairs Clinical Science Research and Development Service (1I21CX000797), and the New Jersey War Related Illness and Injury Study Center. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

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