Erythrocyte deformability reduction in various pediatric hematologic diseases

Abstract

BACKGROUND:

Previously, hemorheology studies using Rheoscan mainly focused on chronic kidney disease, cardiovascular disease, and endocrine disease in adults. The study using LORCA focused on erythrocyte disease. There were no studies using Rheoscan in children.

OBJECTIVE:

We aimed to investigate erythrocyte deformability among various hematologic diseases occurring in children, namely, iron deficiency anemia (IDA), hereditary spherocytosis (HS), immune thrombocytopenia (ITP), and aplastic anemia (AA).

METHODS:

Differences between those with HS, IDA, ITP, AA and healthy controls were compared among 43 patients, comprising 7 patients with HS, 8 patients with IDA, 6 patients with AA, 9 patients with ITP, and 13 healthy controls. Erythrocyte deformability was measured using a microfluidic ektacytometer (RheoScan-D, RheoMeditech, Seoul, Korea). The erythrocyte elongation index (EI) was defined as (L – W)/(L + W), where L and W are the major and minor axes of the ellipse, respectively.

RESULTS:

The EI values of IDA, HS and AA were significantly decreased compared with healthy controls, but those of ITP were similar to healthy controls.

CONCLUSIONS:

This study showed that erythrocyte deformability differed among various hematologic diseases. Further study concerning correlation in relation to the diagnostic and prognostic significance of erythrocyte deformability in hematologic disease is needed.

Keywords

RBC deformability children hereditary spherocytosis iron deficiency anemia aplastic anemia immune thrombocytopenia

1 Introduction

The major organs and tissues of the human body maintain their vitality via blood flow through the blood vessels. Within this process, erythrocytes play a crucial role in the transport of oxygen and carbon dioxide via blood flow through the circulatory system. Erythrocytes are morphologically shaped into centered discs with a diameter of 6–8 μm and are highly variable, allowing them to easily move through 3–5 μm diameter capillaries distributed within organs and tissues. The degree of erythrocyte variability can be expressed through the extent of erythrocyte deformability, which is one rheological feature involved in maintaining human life [1, 2]. Hemorheologic abnormalities have been observed in various clinical situations, including those involving diabetes mellitus, ischemic heart diseases, and hypertension [3 –9].

Previously, hemorheology studies using Rheoscan mainly focused on chronic kidney disease, cardiovascular disease, and endocrine disease in adults [10 –13]. In addition, the study using LORCA focused on erythrocyte disease [14 –16]. There were no studies using Rheoscan in children and on aplastic anemia (AA) and immune thrombocytopenia (ITP).

We undertook this study to investigate hemorrheologic indices in relation to pediatric hematologic diseases.

2 Material and methods

2.1 Patients

Blood samples of pediatric patients diagnosed with hematologic diseases were collected from the Department of Pediatrics, Yeungnam University Hospital. The hematologic diseases investigated included iron deficiency anemia (IDA), hereditary spherocytosis (HS), immune thrombocytopenia (ITP), and aplastic anemia (AA). The differences between patients with HS, IDA, ITP, and AA and healthy controls were compared. The Yeungnam University Medical Center Institutional Review Board (IRB 2014-01-494) approved this study, and it was undertaken in accordance with the Declaration of Helsinki 1975.

2.2 Hematological measurements

The red blood count (RBC) index, including the mean corpuscular volume (MCV in fl), the mean corpuscular hemoglobin concentration (MCHC in g/dL) and hemoglobin concentration, was determined using an automatic counter (ADVIA 2120 Hematology Systems; Bayer HealthCare, Tarrytown, NY, USA).

2.3 RBC deformability measurements

The hemorheologic parameters were analyzed using routine laboratory assays. The erythrocyte deformability (ED), which was quantified using an elongation index (EI) set at a constant shear stress of 3 pascal (Pa), was measured using a microfluidic ektacytometer (RheoScan-D^®; Rheo-Meditech, Seoul, Korea) disposable kit [13], and the other hemorheologic parameters were compared between patients with hematologic diseases and healthy controls. The EI was calculated from the dimensions of the major (L) and minor (W) axes of an ellipsoid at 3 Pa (EI = [L–W]/[L + W]), and expressed as a percentage.

For patients with IDA, blood samples at diagnosis and after iron supplement therapy were examined and compared.

2.4 Statistical analysis

Data are presented as mean±standard deviation (SD). To compare the variables between patients with hematologic diseases and healthy controls, a nonparametric method involving either a Wilcoxon sign rank test or a Mann-Whitney test was used. P-values<0.05 were considered statistically significant. All statistical analysis was performed using SPSS software v25.0 (IBM Corp., Armonk, NY, USA).

3 Results

Forty-three participants were included in the study, comprising 7 patients with HS, 8 patients with IDA, 6 patients with AA, 9 patients with ITP, and 13 healthy controls. The ages of the patients in each category were 8.5±6.1, 12.8±2.8, 12.1±4.2, 3.5±3.7 and 8.5±4.7 years, respectively. Table 1 shows the patient characteristics. Those with HS showed mild anemia, low MCV, and a markedly high reticulocyte count. Those with IDA showed anemia and low MCV, MCH, and MCHC. Those with AA showed a low WBC, hemoglobin (Hb) and platelet count, while those with ITP showed a normal white blood count (WBC) and Hb and a very low platelet count. No differences were found in relation to age and sex before and after iron supplement therapy.

Table 1
Baseline characteristics of the patients

HS IDA AA ITP Normal

Number of patients 7 8 6 9 13

Male, n (%) 4 (57.1) 3 (37.5) 3 (50) 6 (66.7) 6 (46.2)

Age 8.5±6.1 12.8±2.8 12.1±4.2 3.5±3.7 8.5±4.7

WBC (K/μL) 7.61±2.29 5.76±1.50 2.52±0.87 6.24±3.08 7.05±1.55

Hb (g/dL) 12.1±2.5 8.4±1.8 8.1±1.9 12.2±0.6 13.4±1.1

Platelet (K/μL) 435±169 335±79 20±9 24±16 316±83

MCV (fL) 75.5±6.0 67.5±9.9 95.3±10.6 76.9±3.2 80.6±4.0

MCH (pg) 27.9±2.2 20.1±4.4 33.6±3.9 26.5±1.4 28.2±1.6

MCHC (g/dL) 36.9±0.8 29.6±2.3 29.9±13.3 34.4±0.9 35.0±0.9

Reti count (%) 8.03±5.38 3.88±3.21 5.96±2.91 1.63±0.53 1.50±0.66

	HS	IDA	AA	ITP	Normal
Number of patients	7	8	6	9	13
Male, n (%)	4 (57.1)	3 (37.5)	3 (50)	6 (66.7)	6 (46.2)
Age	8.5±6.1	12.8±2.8	12.1±4.2	3.5±3.7	8.5±4.7
WBC (K/μL)	7.61±2.29	5.76±1.50	2.52±0.87	6.24±3.08	7.05±1.55
Hb (g/dL)	12.1±2.5	8.4±1.8	8.1±1.9	12.2±0.6	13.4±1.1
Platelet (K/μL)	435±169	335±79	20±9	24±16	316±83
MCV (fL)	75.5±6.0	67.5±9.9	95.3±10.6	76.9±3.2	80.6±4.0
MCH (pg)	27.9±2.2	20.1±4.4	33.6±3.9	26.5±1.4	28.2±1.6
MCHC (g/dL)	36.9±0.8	29.6±2.3	29.9±13.3	34.4±0.9	35.0±0.9
Reti count (%)	8.03±5.38	3.88±3.21	5.96±2.91	1.63±0.53	1.50±0.66

AA, aplastic anemia; Hb, hemoglobin; HS, Hereditary spherocytosis; IDA, iron deficiency anemia; ITP, immune thrombocytopenia; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; Reti count, reticulocyte count.

The Hb, MCV, MCH, and MCHC of patients with IDA decreased significantly compared to those of healthy controls, and the RBC index, comprising Hb, MCV, MCH and MCHC, increased significantly after iron supplement therapy (Table 2).

Table 2

RBC index during iron supplement therapy in patients with IDA compared to healthy controls

	Before iron therapy	After iron therapy	Healthy controls	p-value^a	p-value^b
Hb	8.4±1.8	10.8±1.9	13.4±1.1	0.008	<0.001
MCV	67.5±9.9	74.1±12.8	80.6±4.0	0.008	0.002
MCH	20.1±4.4	22.9±4.5	28.2±1.6	0.008	0.001
MCHC	29.6±2.3	30.8±1.5	35.0±0.9	0.008	<0.001

Hb, hemoglobin; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume. ^aResults obtained before vs after iron supplement therapy, using a Wilcoxon signed rank test. ^bResults obtained before iron supplement therapy vs healthy controls, using a Mann-Whitney test.

The EI at 3 Pa was significantly lower than normal in patients prior to iron supplement therapy (0.226±0.028 vs 0.308±0.013, p < 0.001) (Fig. 1). The EI at 3 Pa was significantly increased after iron supplement therapy in patients with IDA (0.267±0.031 vs 0.308±0.013, p < 0.001) (Fig. 1).

Fig.1

Comparison of the elongation index at 3 Pa between healthy controls and A (patients before and after iron supplement therapy, B (patients with aplastic anemia), C (patients with hereditary spherocytosis, and D (patients with immune thrombocytopenia).

The EI at 3 Pa was significantly lower than normal in patients with AA (Fig. 1(B)). There was no significant difference in EI at 3 Pa in patients with HS or ITP. However, when analyzing the EI according to pressure, it was found to be lower than 7 Pa in patients with HS compared to that in the healthy controls (Table 3).

Table 3

Comparison of the elongation index between patients with hereditary spherocytosis (HS) and healthy controls according to pressure

Pa	HS	Healthy controls	p-value
0.5	0.062±0.052	0.026±0.042	0.115
1	0.176±0.026	0.073±0.306	0.067
1.5	0.228±0.031	0.215±0.012	0.157
2	0.263±0.032	0.254±0.012	0.393
2.5	0.288±0.032	0.284±0.013	0.485
3	0.308±0.031	0.308±0.013	1.000
4	0.336±0.031	0.344±0.013	0.643
5	0.356±0.031	0.370±0.013	0.183
6	0.371±0.032	0.390±0.013	0.056
7	0.383±0.033	0.406±0.013	0.030
8	0.392±0.034	0.419±0.013	0.024
10	0.407±0.035	0.440±0.012	0.011
12	0.418±0.036	0.455±0.012	0.005
15	0.429±0.037	0.473±0.011	0.003
17	0.435±0.038	0.482±0.010	<0.001
20	0.442±0.038	0.492±0.010	<0.001

4 Discussion

This study was the first to use a microfluidic ektacytometer (RheoScan-D) in investigating hematologic diseases in children. Previously, studies of RBC deformability in relation to various hematologic diseases have been conducted using a laser optical rotational red cell analyzer (LORCA) ektacytometer [14 , 17].

Halis et al. studied changes in hemorrheologic parameters according to iron supplementation in children with IDA. Although IDA resulted in a decrement in RBC deformability, aggregation, plasma, and whole blood viscosities, these parameters returned to control values after iron supplementation therapy. Serum ferritin levels and hematological parameters (Hb, MCV, MCH, MCHC) that were lower in patients with IDA were also found to be increased after treatment. A laser-assisted ektacytometer (LORCA; RR Mechatronics, Hoorn, The Netherlands) was used in that study and the EI values were analyzed using the results obtained at 1.69 Pa [16].

Zaninoni et al. conducted a study using a LORCA MaxSis to diagnose RBC disease in 202 patients [14]. Patients with HS showed altered Osmoscan curves, with a decreased EI maximum (max) and right shifted Omin, while patients with hereditary elliptocytosis (HE) displayed a trapezoidal curve and decreased EI max. However, their results should be interpreted with caution as other factors (i.e., splenectomy or coexistent diseases) may have interfered with the analysis. Additional tests or molecular investigations would therefore be needed to confirm those results.

Our study used RheoScan-D equipment and analyzed the EI values at 3 Pa. At the time of diagnosis of IDA, the EI values were decreased. As iron supplementation increased, the RBC index (Hb, MCV, MCH, MCHC) and the EI values increased.

Llaudet-Planas et al. analyzed patients with HS, HE and hereditary xerocytosis compared to healthy controls using osmotic gradient ektacytometry to determine optimal cutoff values in relation to HS [18]. They used a LORCA MaxSis instrument, increasing the osmotic gradient from 80 mOsmol/L to 500 mOsmol/L under a shear stress of 30 mPa. Several hemorheologic parameters (EImin, Omin, EImax, Omax, Ohyper, and EIhyper) were measured. In their study, the best diagnostic criteria for HS were found to be a combination of a decreased minimal elongation index up to 3% and an increased minimal osmolality point up to 5.2% when compared to the mean of the healthy controls. Osmotic gradient ektacytometry indicated a sensitivity of 93.85% and a specificity of 98.38% for the diagnosis of HS.

In our study, no differences were found in EI values between patients with HS and the healthy controls up to 6 Pa in shear stress. However, above 7 Pa, the EI values of patients with HS were lower than those of the healthy controls.

A decreased EI at 3 Pa in patients with AA has not been reported in previous studies. Shin et al. reported a decrease in deformability in patients with diabetes mellitus with chronic renal failure compared to that in healthy patients, and that, as patients’ renal function decreased, an increased impairment in RBC deformability was found [13].

Park et al. reported that EI values in patients with type 2 diabetes mellitus were lower in those without acute myocardial infarction (AMI) compared to those in patients with AMI [10]. The gradual exacerbation of ED due to glucose tolerance damage is already known to be an indicator of microangiopathy [19 –21].

We have previously reported that erythrocytes exposed to oxidative stress have decreased deformability. We concluded that a change of hemoglobin to methemoglobin affects ED [12].

In another study, we evaluated the hemorheological properties, the degree of lipid peroxidation and the oxidative susceptibility of irradiated RBCs. Exposure to gamma rays significantly increased the MCV and lipid peroxidation. Changes in RBC deformability were more prominent in irradiated RBCs than in non-irradiated RBCs also under conditions of oxidative stress. That study indicated that exposure to gamma rays reduces RBC deformability during storage and irradiated RBCs seem to be susceptible to oxidative stress [11].

The mechanism by which RBC deformability decreases in patients with AA is not clear. AA is characterized by bone marrow (BM) hypocellularity, resulting in peripheral cytopenia. An antigen-driven and likely auto-immune dysregulated T-cell homeostasis results in hematopoietic stem cell injury, which ultimately contributes to the pathogenesis of AA in relation to changes to bone marrow hematopoietic stem cells and the surrounding microenvironment [22, 23]. Therefore, this process appears to affect RBC production, reducing deformability. In addition, microangiopathy due to the effects of chronic disease also appears to affect deformability.

5 Conclusions

This is the first study to study RBC deformability using RheoScan-D equipment in relation to various pediatric hematologic diseases.

As with other existing studies, it was found that ED was significantly reduced in patients with AA, HS and IDA.

ITP was found not to affect ED despite circulating antibodies to platelets;

Further studies with more patients may facilitate improved diagnosis, severity assessment and prognostic findings in terms of RBC deformability concerning pediatric hematologic diseases.

Acknowledgments

Source of funding

This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (No. NRF-2017R1D1A1A02019498).

Conflict of interest

The authors report no conflicts of interest.

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